Therapeutic Compounds for Red Blood Cell-Mediated Delivery of an Active Pharmaceutical Ingredient to a Target Cell

ABSTRACT

Therapeutic compounds for red blood cell-mediated delivery of an active pharmaceutical ingredient to a target cell are described. The therapeutic compounds are configured to bind CD47 on the surface of a red blood cell and to be subsequently transferred to CD47 on the surface of the target cell, the therapeutic compound ultimately being internalized by the target cell via endocytosis. The target cell may be a cancer cell, a virus-infected cell, or a fibrotic cell.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims priority from U.S. Provisional Application No. 63/281,370, filed Nov. 19, 2021, and U.S. Provisional Application No. 63/392,323, filed Jul. 26, 2022, each of which is incorporated herein by reference in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in XML file format and is hereby incorporated by reference in its entirety. Said XML copy, created on Nov. 17, 2022, is named 4813_1004_SL.xml and is 4,562,889 bytes in size.

TECHNICAL FIELD

The present invention relates generally to a therapeutic compound configured to bind CD47, and more particularly to such a compound configured to bind CD47 on the surface of a red blood cell and to be subsequently transferred to CD47 on the surface of a target cell, the therapeutic compound ultimately being internalized by the target cell via endocytosis.

BACKGROUND ART

Cluster of differentiation 47 (“CD47”), an integrin-associated protein, is a multi-spanning plasma membrane protein involved in the processes inhibiting clearance by phagocytes or neutrophil motility. Signal-regulatory protein alpha (“SIRPα”), a transmembrane protein expressed by innate immune cells such as macrophages and dendritic cells, is the main receptor of CD47. The binding of SIRPα to CD47 triggers SIRPα inhibitory signals, which act as “don't eat me” signals to recipient macrophages, preventing their phagocytic activation. Thus, the SIRPα-CD47 interaction functions as a negative checkpoint for innate and subsequent adaptive immunity. Other proteins, such as signal regulatory protein gamma (“SIRPγ”) and thrombospondin-1 (“TSP-1”) can also bind CD47, thereby inhibiting aspects of immune response.

Mammalian cells typically express low levels of CD47 to protect them from phagocytosis. However, cancer cells overexpress CD47 as an evasion mechanism to escape immune surveillance and attack by phagocytic cells. Several human solid tumors overexpress CD47, i.e., the cells of these solid tumors express more CD47 than normal cells on average (Willingham et al. PNAS 109(17):6662-6667 (2012), which is hereby incorporated by reference herein in its entirety). CD47 has thus emerged as a promising new therapeutic target for cancer immunotherapy (Willingham et al. PNAS 109(17):6662-6667 (2012); Weiskopf, Eur. J. Cancer 76:100-109 (2017); Weiskopf et al. J Clin Invest 126(7):2610-2620 (2016), each of which is hereby incorporated by reference herein in its entirety).

In addition, virus-infected cells also express high levels of CD47. These virus-infected cells include cells infected with SARS-CoV-2, the virus that causes COVID-19 (Cham et al. Cell Rep 14; 31(2):107494 (2020) doi:10.1016/j.celrep.2020.03.058 and McLaughlin et al. bioRxiv 2021.03.01.433404 (2021) doi:10.1101/2021.03.01.433404, each of which is hereby incorporated by reference herein in its entirety). Blockade of CD47 inhibitory signaling has been demonstrated to enhance innate and adaptive immune responses to viral infection.

Moreover, increased CD47 expression has been observed in fibrotic fibroblasts and blocking CD47 reverses fibrosis by increasing phagocytosis of profibrotic fibroblasts and by eliminating suppressive effects on adaptive immunity (Cui et al. Nat Commun 11:2795 (2020); Wernig et al. PNAS 2017; 114(18):4757-62; Boyd J Cyst Fibros Suppl 1:S54-S59 (2020); Lerbs et al. JCI Insight 2020; 5(16):e140458 (2020), each of which is hereby incorporated by reference herein in its entirety).

CD47, therefore, offers a promising target for the treatment of cancers, viral infections, as well as fibrotic diseases, such as cystic fibrosis.

SUMMARY OF THE EMBODIMENTS

In accordance with one embodiment of the invention, a therapeutic compound for RBC-mediated delivery in a mammalian subject to a target cell expressing CD47, the therapeutic compound comprising: a CD47-binding protein conjugated to an active pharmaceutical ingredient (“API”) so as to form a conjugate; wherein the CD47-binding protein is selected from the group consisting of wild type SIRPα (SEQ ID NO: 1), vSIRPα (SEQ ID NO: 3), wild type thrombospondin-1 (TSP-1) (SEQ ID NO: 7), wild type SIRPγ (SEQ ID NO: 4), vSIRPγ-1 (SEQ ID NO: 5), vSIRPγ-2 (SEQ ID NO: 6), ALX148 (SEQ ID NO: 962), TTI-661 (SEQ ID NO: 963), TTI-662 (SEQ ID NO: 964), a homolog of any of the foregoing, and combinations thereof, and is configured to bind the conjugate to CD47 of a red blood cell of the subject so as to enable transport of the conjugate, through the subject's circulatory system, to the target cell, so that (i) the CD47-binding protein, being configured to bind the conjugate to the CD47 of the red blood cell, binds the CD47 of the target cell, thus transferring the conjugate from the red blood cell to the target cell so as to form a conjugate-CD47 complex on the target cell, thereby blocking CD47 and inhibiting CD47 activity as an immune escape mechanism of the target cell, and (ii) the conjugate is taken up by the target cell via endocytosis of the conjugate-CD47 complex, thereby further inhibiting the immune escape mechanism of the target cell and delivering the API into the target cell. The mammalian subject may be a human.

In accordance with another embodiment of the invention, a therapeutic compound for RBC-mediated delivery in a mammalian subject to a target cell expressing CD47, the therapeutic compound comprising: a CD47-binding protein conjugated to an API so as to form a conjugate; wherein the CD47-binding protein is selected from the group consisting of wild type thrombospondin-1 (TSP-1) (SEQ ID NO: 7), wild type SIRPγ (SEQ ID NO: 4), vSIRPγ-1 (SEQ ID NO: 5), vSIRPγ-2 (SEQ ID NO: 6), ALX148 (SEQ ID NO: 962), TTI-661 (SEQ ID NO: 963), TTI-662 (SEQ ID NO: 964), a homolog of any of the foregoing, and combinations thereof, and is configured to bind the conjugate to CD47 of a red blood cell of the subject so as to enable transport of the conjugate, through the subject's circulatory system, to the target cell, so that (i) the CD47-binding protein, being configured to bind the conjugate to the CD47 of the red blood cell, binds the CD47 of the target cell, thus transferring the conjugate from the red blood cell to the target cell so as to form a conjugate-CD47 complex on the target cell, thereby blocking CD47 and inhibiting CD47 activity as an immune escape mechanism of the target cell, and (ii) the conjugate is taken up by the target cell via endocytosis of the conjugate-CD47 complex, thereby further inhibiting the immune escape mechanism of the target cell and delivering the API into the target cell. The mammalian subject may be a human.

In accordance with an embodiment of the invention, a therapeutic compound for RBC-mediated delivery in a mammalian subject to a target cell expressing CD47, the therapeutic compound comprising: a CD47-binding protein conjugated to an API so as to form a conjugate; wherein the CD47-binding protein is an anti-CD47 antibody, the anti-CD47 antibody comprising: (a) a heavy chain variable region including complementarity determining regions CDR1, CDR2, and CDR3 comprising SEQ ID NO: 932, SEQ ID NO: 933, and SEQ ID NO: 934, respectively, and a light chain variable region including complementarity determining regions CDR1, CDR2, and CDR3 comprising SEQ ID NO: 935, SEQ ID NO: 936, and SEQ ID NO: 937, respectively; (b) a heavy chain variable region including complementarity determining regions CDR1, CDR2, and CDR3 comprising SEQ ID NO: 940, SEQ ID NO: 941, and SEQ ID NO: 942, respectively, and a light chain variable region including complementarity determining regions CDR1, CDR2, and CDR3 comprising SEQ ID NO: 943, SEQ ID NO: 944, and SEQ ID NO: 945, respectively; (c) a heavy chain variable region including complementarity determining regions CDR1, CDR2, and CDR3 comprising SEQ ID NO: 948, SEQ ID NO: 949, and SEQ ID NO: 950, respectively, and a light chain variable region including complementarity determining regions CDR1, CDR2, and CDR3 comprising SEQ ID NO: 951, SEQ ID NO: 952, and SEQ ID NO: 953, respectively; or (d) a heavy chain variable region including complementarity determining regions CDR1, CDR2, and CDR3 comprising SEQ ID NO: 956, SEQ ID NO: 957, and SEQ ID NO: 958, respectively, and a light chain variable region including complementarity determining regions CDR1, CDR2, and CDR3 comprising SEQ ID NO: 959, SEQ ID NO: 960, and SEQ ID NO: 961, respectively; and being configured to bind the conjugate to CD47 of a red blood cell of the subject so as to enable transport of the conjugate, through the subject's circulatory system, to the target cell, so that (i) the CD47-binding protein, being configured to bind the conjugate to the CD47 of the red blood cell, binds the CD47 of the target cell, thus transferring the conjugate from the red blood cell to the target cell so as to form a conjugate-CD47 complex on the target cell, thereby blocking CD47 and inhibiting CD47 activity as an immune escape mechanism of the target cell, and (ii) the conjugate is taken up by the target cell via endocytosis of the conjugate-CD47 complex, thereby further inhibiting the immune escape mechanism of the target cell and delivering the API into the target cell. The mammalian subject may be a human.

The CD47-binding protein may be conjugated to the API by a bond selected from the group consisting of a covalent bond, a hydrogen bond, an ionic bond, a van der Waals interaction, and combinations thereof. The CD47-binding protein may be conjugated to the API by a linker and the linker may be cleavable. The linker may be configured to be cleaved by a lysosomal degradative enzyme.

In some embodiments, the API is selected from the group consisting of RNA, DNA, an RNA derivative, a DNA derivative, a protein, and a small molecule. The RNA may be selected from the group consisting of siRNA, shRNA, miRNA, antimiR, and mRNA.

The target cell may be a cell selected from the group consisting of a cancer cell, a virus infected cell, a fibrotic cell, and combinations thereof. In some embodiments, the target cell is a cancer cell. In some embodiments, the target cell is a virus-infected cell. In some embodiments, the target cell is a fibrotic cell.

In some embodiments, the cancer cell is in a tumor attributable to a cancer selected from the group consisting of brain tumor, spinal cord tumor, retinoblastoma, oral cancer, nasal cavity cancer, paranasal sinus cancer, pharyngeal cancer, laryngeal cancer, neck cancer, head and neck cancer, melanoma, skin cancer, breast cancer, thyroid cancer, malignant adrenal tumor, endocrine cancer, lung cancer, pleural tumor, respiratory tract cancer, esophageal cancer, stomach cancer, small intestine cancer, colon cancer, anal cancer, liver cancer, biliary tract cancer, pancreatic cancer, kidney cancer, bladder cancer, prostate cancer, testicular cancer, penile cancer, cervical cancer, endometrial cancer, choriocarcinoma, ovarian cancer, blood cancer including acute/chronic leukemia, malignant lymphoma and multiple myeloma, bone tumor, soft tissue tumor, childhood leukemia, and childhood cancer.

In some embodiments, the cancer cell is attributable to a cancer selected from the group consisting of ovarian serous cystadenocarcinoma, lung adenocarcinoma, cervical and endocervical cancer, head and neck squamous cell carcinoma, thyroid carcinoma, uterine corpus endometrioid carcinoma, prostate adenocarcinoma, mesothelioma, diffuse large B-cell lymphoma, acute leukemia, lung squamous cell carcinoma, acute lymphoblastic leukemia, esophageal carcinoma, myxofibrosarcoma, pancreatic adenocarcinoma, rectum adenocarcinoma, colon adenocarcinoma, acute megakaryoblastic leukemia, breast invasive carcinoma, stomach adenocarcinoma, bladder urothelial carcinoma, cholangiocarcinoma, leukemia, thymic carcinoma, leiomyosarcoma, thymoma, undifferentiated pleomorphic sarcoma, uterine carcinosarcoma, acute myeloid leukemia, glioblastoma multiforme, sarcoma, skin cutaneous melanoma, kidney clear cell carcinoma, dedifferentiated liposarcoma, lymphoma, retinoblastoma, neuroblastoma, osteosarcoma, juvenile myelomonocytic leukemia, gastrointestinal stromal tumor, dysembryoplatic neuroepithelial tumor, adrenocortical cancer, acute leukemia of ambiguous lineage, pheochromocytoma and paraganglioma, glioma, testicular germ cell tumor, supratentorial embryonal tumor NOS, neurofibroma, kidney papillary cell carcinoma, hepatocellular carcinoma, kidney chromophobe, malignant peripheral nerve sheath tumor, ependymoma, adrenocortical carcinoma, nasopharyngeal carcinoma, spindle cells/sclerosing rhabdomyosarcoma, melanoma, choroid plexus carcinoma, undifferentiated spindle cell carcinoma, myoepithelial carcinoma, alveolar rhabdomyosarcoma, rhabdomyosarcoma, atypical teratoid/rhabdoid tumor, desmoplastic small round cell tumor, fibromatosis, synovial sarcoma, wilms tumor, myofibromytosis, fibrolamellar hepatocellular carcinoma, undifferentiated sarcoma NOS, embryonal rhabdomyosarcoma, uveal melanoma, Ewing sarcoma, hepatoblastoma, infantile fibrosarcoma, INI-deficient soft tissue sarcoma NOA, undifferentiated hepatic sarcoma, and medulloblastoma.

In some embodiments, the virus-infected cell is infected with a SARS-CoV-2 virus. In some embodiments, the fibrotic cell is associated with cystic fibrosis.

In some embodiments, the API is siRNA, the siRNA being a double-stranded RNA molecule including an antisense RNA strand and a sense RNA strand, wherein: (a) the antisense RNA strand is 19-29 nucleotides in length and is complementary to contiguous nucleotides in a target mammalian mRNA sequence, the mRNA sequence being selected from the group consisting of SEQ ID NO: 8-747 and 771-824, (b) the sense RNA strand is 19-29 nucleotides in length and is complementary to 14-29 nucleotides from the antisense RNA strand, and (c) the double stranded RNA molecule has a double stranded region of 14-29 nucleotides in length and a 3′ overhang region of 0-5 nucleotides in length. In other embodiments, the mRNA sequence is selected from the group consisting of SEQ ID NO: 22-747 and 771-824. In some embodiments, the mRNA sequence is selected from the group consisting of SEQ ID NO: 22-37. In other embodiments, the mRNA sequence is selected from the group consisting of SEQ ID NO: 38-39. In some embodiments, the mRNA sequence is selected from the group consisting of SEQ ID NO: 40-43. In other embodiments, the mRNA sequence is selected from the group consisting of SEQ ID NO: 44-51.

In some embodiments, the API is siRNA, the siRNA being a double-stranded RNA molecule including an antisense RNA strand and a sense RNA strand, wherein: (a) the antisense RNA strand is 19-29 nucleotides in length and is complementary to contiguous nucleotides in a target mammalian mRNA sequence, the mRNA sequence being selected from the group consisting of SEQ ID NO: 8-21, 482-486, and 748-765, (b) the sense RNA strand is 19-29 nucleotides in length and is complementary to 14-29 nucleotides from the antisense RNA strand, and (c) the double stranded RNA molecule has a double stranded region of 14-29 nucleotides in length and a 3′ overhang region of 0-5 nucleotides in length. In other embodiments, the mRNA sequence is selected from the group consisting of SEQ ID NO: 482-486 and 748-765.

In some embodiments, the API is siRNA, the siRNA being a double-stranded RNA molecule including an antisense RNA strand and a sense RNA strand, wherein: (a) the antisense RNA strand is 19-29 nucleotides in length and is complementary to contiguous nucleotides in a target mammalian mRNA sequence, the mRNA sequence being selected from the group consisting of SEQ ID NO: 8-21, 40-43, and 766-770, (b) the sense RNA strand is 19-29 nucleotides in length and is complementary to 14-29 nucleotides from the antisense RNA strand, and (c) the double stranded RNA molecule has a double stranded region of 14-29 nucleotides in length and a 3′ overhang region of 0-5 nucleotides in length. In other embodiments, the mRNA sequence is selected from the group consisting of SEQ ID NO: 40-43 and 766-770.

The API is shRNA, the shRNA being a single-stranded RNA molecule of 44-71 nucleotides in length, and having, in a 5′ to 3′ direction: a first region of 19-29 nucleotides at the 5′ end of the single-stranded RNA molecule, the first region having a first sequence; a second region of 4-11 nucleotides directly adjacent to the first region, the second region having a second sequence; a third region of 19-29 nucleotides directly adjacent to the second region, the third region having a third sequence; and a fourth region of 2 nucleotides at the 3′ end of the single-stranded RNA molecule, directly adjacent to the third region, the fourth region having a fourth sequence, wherein: (a) the first region has the same number of nucleotides as the third region, (b) the third sequence is the reverse-complement of the first sequence, (c) the third region is complementary to contiguous nucleotides in a target mammalian mRNA sequence, the mRNA sequence being selected from the group consisting of SEQ ID NO: 8-747 and 771-824, and (d) the single-stranded RNA molecule is configured to form a stem loop structure, the first region base pairing with the third region to form a stem, the second region forming a loop, and the fourth region forming a 3′ overhang. In other embodiments, the mRNA sequence is selected from the group consisting of SEQ ID NO: 22-747 and 771-824. In some embodiments, the mRNA sequence is selected from the group consisting of SEQ ID NO: 22-37. In other embodiments, the mRNA sequence is selected from the group consisting of SEQ ID NO: 38-39. In some embodiments, the mRNA sequence is selected from the group consisting of SEQ ID NO: 40-43. In other embodiments, the mRNA sequence is selected from the group consisting of SEQ ID NO: 44-51.

In some embodiments, the API is shRNA, the shRNA being a single-stranded RNA molecule of 44-71 nucleotides in length, and having, in a 5′ to 3′ direction: a first region of 19-29 nucleotides at the 5′ end of the single-stranded RNA molecule, the first region having a first sequence; a second region of 4-11 nucleotides directly adjacent to the first region, the second region having a second sequence; a third region of 19-29 nucleotides directly adjacent to the second region, the third region having a third sequence; and a fourth region of 2 nucleotides at the 3′ end of the single-stranded RNA molecule, directly adjacent to the third region, the fourth region having a fourth sequence, wherein: (a) the first region has the same number of nucleotides as the third region, (b) the third sequence is the reverse-complement of the first sequence, (c) the third region is complementary to contiguous nucleotides in a target mammalian mRNA sequence, the mRNA sequence being selected from the group consisting of SEQ ID NO: 8-21, 482-486, and 748-765, and (d) the single-stranded RNA molecule is configured to form a stem loop structure, the first region base pairing with the third region to form a stem, the second region forming a loop, and the fourth region forming a 3′ overhang. In other embodiments, the mRNA sequence is selected from the group consisting of SEQ ID NO: 482-486 and 748-765.

In some embodiments, the API is shRNA, the shRNA being a single-stranded RNA molecule of 44-71 nucleotides in length, and having, in a 5′ to 3′ direction: a first region of 19-29 nucleotides at the 5′ end of the single-stranded RNA molecule, the first region having a first sequence; a second region of 4-11 nucleotides directly adjacent to the first region, the second region having a second sequence; a third region of 19-29 nucleotides directly adjacent to the second region, the third region having a third sequence; and a fourth region of 2 nucleotides at the 3′ end of the single-stranded RNA molecule, directly adjacent to the third region, the fourth region having a fourth sequence, wherein: (a) the first region has the same number of nucleotides as the third region, (b) the third sequence is the reverse-complement of the first sequence, (c) the third region is complementary to contiguous nucleotides in a target mammalian mRNA sequence, the mRNA sequence being selected from the group consisting of SEQ ID NO: 8-21, 40-43, and 766-770, and (d) the single-stranded RNA molecule is configured to form a stem loop structure, the first region base pairing with the third region to form a stem, the second region forming a loop, and the fourth region forming a 3′ overhang. In other embodiments, the mRNA sequence is selected from the group consisting of SEQ ID NO: 40-43 and 766-770.

In some embodiments, the API is an miRNA selected from the group consisting of SEQ ID NO: 825-844, 849-851, 853, 855, 857, 864, 865, and 867-883.

In some embodiments, the API is an antimiR, the antimiR being a single-stranded nucleic acid molecule of 12-25 nucleotides in length, the antimiR having a sequence of 12-25 contiguous nucleotides that is complementary to contiguous nucleotides in a target mature miRNA product sequence, the mature miRNA product sequence being selected from the group consisting of SEQ ID NO: 884-908, wherein the contiguous nucleotides in the mature miRNA product sequence includes, in a 5′ to 3′ direction, nucleotides 2 to 8 of the mature miRNA product sequence.

In some embodiments, the API is a small molecule selected from the group consisting of methotrexate; doxorubicin; vinca alkaloids; camptothecin analogues; microtubule-disrupting agents such as auristatins (e.g., MMAE and MMAF) and maytansinoids (e.g., DM1 and DM4); and DNA-damaging agents such as DNA topoisomerase I inhibitors (e.g., SN-38 and exatecan), double-strand break agents (e.g., calicheamicin), cross-linkers (e.g., pyrrolobenzodiazepine dimer-PBD), and alkylators (e.g., duocarmycin and indolinobenzodiazepine dimer-IGN).

In some embodiments, the API is a protein, the protein having an amino acid sequence selected from the group consisting of SEQ ID NO: 909-929 and homologs thereof. In other embodiments, the protein consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 909-929 and homologs thereof.

In some embodiments, the API is an mRNA encoding an amino acid sequence selected from the group consisting of SEQ ID NO: 909-929 and homologs thereof, the mRNA being configured to be translated in the target cell to produce a protein comprising the amino acid sequence. In other embodiments, the mRNA is configured to be translated in the target cell to produce a protein consisting of the amino acid sequence. In some embodiments, the mRNA is codon optimized.

In accordance with one embodiment of the invention, a method of treating cancer in a mammalian subject in need thereof, the method comprising administering a therapeutically effective amount of a therapeutic compound described herein. The mammalian subject may be a human.

In accordance with another embodiment of the invention, a method of treating viral infection in a mammalian subject in need thereof, the method comprising administering a therapeutically effective amount of a therapeutic compound described herein. The mammalian subject may be a human.

In accordance with an embodiment of the invention, a method of treating fibrotic disease in a mammalian subject in need thereof, the method comprising administering a therapeutically effective amount of a therapeutic compound described herein. The mammalian subject may be a human.

In accordance with another embodiment of the invention, a pharmaceutical composition comprising a therapeutic compound described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

The foregoing features of embodiments will be more readily understood by reference to the following detailed description, taken with reference to the accompanying drawings, in which:

FIG. 1A illustrates a FAM-tagged vSIRPα-siRNA conjugate in accordance with embodiments of the invention. FIG. 1B is an illustration of the incubation of red blood cells with the FAM-tagged vSIRPα-siRNA conjugate of FIG. 1A. FIG. 1C shows fluorescence microscopy images of red blood cells that were not incubated with the FAM-tagged vSIRPα-siRNA conjugate shown in FIG. 1A (left) and red blood cells that were incubated with the FAM-tagged vSIRPα-siRNA conjugate shown in FIG. 1A (right).

FIG. 2A shows flow cytometry results of CaCO2 cells before and after being incubated with red blood cells bound with FAM-tagged vSIRPα-siRNA, in accordance with embodiments of the invention. FIG. 2B shows flow cytometry results of CT26.CL25 cells before and after being incubated with red blood cells bound with FAM-tagged vSIRPα-siRNA.

FIG. 3A shows flow cytometry results of CaCO2 cells before and after being incubated with red blood cells bound with an Alexa Fluor® 647 anti-mouse CD47 monoclonal antibody, in accordance with embodiments of the invention. FIG. 3B shows flow cytometry results of CT26.CL25 cells before and after being incubated with red blood cells bound with an Alexa Fluor® 647 anti-mouse CD47 monoclonal antibody.

FIG. 4 shows flow cytometry results of CaCO2 cells and CT26.CL25 cells before (“unstained”) and after being incubated with red blood cells bound with FAM-tagged vSIRPα-siRNA, in accordance with embodiments of the invention.

FIG. 5 shows flow cytometry results of CaCO2 cells and CT26.CL25 cells before (“unstained”) and after being incubated with red blood cells bound with Cy5.5-labeled vSIRPα, in accordance with embodiments of the invention.

FIG. 6 shows flow cytometry results of CT26.CL25 cells before (“unstained”) and after being incubated with red blood cells bound with an Alexa Fluor® 647 anti-mouse CD47 monoclonal antibody, in accordance with embodiments of the invention.

FIG. 7 shows flow cytometry results of CT26.CL25 cells before (“unstained”) and after being incubated with red blood cells bound with a CD47mAb-miR21-Cy5 conjugate, in accordance with embodiments of the invention.

FIG. 8 shows flow cytometry results of CaCO2 cells and CT26.CL25 cells before (“unstained”) and after being incubated with red blood cells bound with Cy5.5-labeled murine thrombospondin-1, in accordance with embodiments of the invention.

FIG. 9 shows red blood cells isolated from an untreated mouse, a mouse injected with a fluorescently labeled siRNA conjugate, and a mouse injected with a fluorescently labeled vSIRPα-siRNA conjugate, in accordance with embodiments of the invention,

DETAILED DESCRIPTION OF SPECIFIC EMBODIMENTS

As used in this description and the accompanying claims, the following terms shall have the meanings indicated, unless the context otherwise requires:

The terms “a” and “an” and “the” and similar reference used in the context of describing the invention (especially in the context of the claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention

A “set” includes at least one member.

The term “mammal,” and the like, refers to any animal species of the Mammalian class. Examples of mammals include humans; laboratory animals such as rats, mice, simians and guinea pigs; domestic animals such as rabbits, cattle, sheep, goats, cats, dogs, horses, pigs, and the like.

“Active pharmaceutical ingredient,” “API,” and the like, means the non-CD47-binding protein portion and the non-linker portion of a therapeutic compound, in accordance with embodiments of the invention, that is biologically active. Suitable active pharmaceutical ingredients (“APIs”) include RNA (siRNA, miRNA, shRNA, and mRNA), DNA, antimiR oligonucleotides (RNA, DNA, and derivatives thereof), RNA and DNA derivatives (including, but not limited to, modified RNA and DNA comprising a modified backbone, sugar, and/or base), proteins, and small molecules.

As used herein, a “homolog” of a given protein, and the like, shall mean a protein having at least 95% sequence identity with the given protein.

“Complementarity,” as used herein regarding nucleic acid sequences, refers to the ability of a nucleic acid to forms hydrogen bonds with another nucleic acid sequence by Watson-Crick base pairing or wobble base pairing. A percent complementarity indicates the percentage of nucleotides in a nucleic acid molecule that can form hydrogen bonds (e.g., Watson-Crick base pairing) with the nucleotides of a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 being 50%, 60%, 70%, 80%, 90%, and 100% complementarity). “Perfectly complementary,” and the like, means that all the contiguous nucleotides of a nucleic acid sequence will hydrogen bond with the same number of contiguous nucleotides in a second nucleic acid sequence (i.e., the nucleic acid sequence has 100% complementarity). “Complementary,” as used herein without further qualification, means that contiguous nucleotides of a nucleic acid sequence has a percent complementarity with contiguous nucleotides of a second nucleic acid sequence that is selected from the group consisting of 95%, 96%, 97%, 98%, 99%, and 100% complementarity over a region of 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or more nucleotides of the nucleic acid sequence. For example, a nucleic acid sequence that is 19 nucleotides in length and complementary to 14 nucleotides of a second nucleic acid sequence means that 14 contiguous nucleotides of the nucleic acid sequence has a percent complementarity with contiguous nucleotides of the second nucleic acid sequence that is selected from the group consisting of 95%, 96%, 97%, 98%, 99%, and 100% complementarity. A nucleic acid sequence that is 19 nucleotides in length and complementary to contiguous nucleotides in a second nucleic acid sequence means that 19 contiguous nucleotides of the nucleic acid sequence has a percent complementarity with contiguous nucleotides of the second nucleic acid sequence that is selected from the group consisting of 95%, 96%, 97%, 98%, 99%, and 100% complementarity.

“Codon-optimized” means that the coding sequence of an mRNA transcript contains the most or second most preferred codon, for the species of a given target cell/host cell, for at least 60% of the codons of the coding sequence such that the codon-optimized sequence is more efficiently translated in the target cell/host cell relative to a non-optimized sequence.

The term “antibody” refers to an immunoglobulin molecule that is typically composed of two identical pairs of polypeptide chains, each pair having one “heavy” (H) chain and one “light” (L) chain. Human light chains are classified as kappa (κ) and lambda (k). Heavy chains are classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. Each heavy chain is comprised of a heavy chain variable region (VH) and a heavy chain constant region. The heavy chain constant regions of IgD, IgG, and IgA are comprised of three domains, CH1, CH2 and CH3, and the heavy chain constant regions of IgM and IgE are comprised of four domains, CH1, CH2, CH3, and CH4. Each light chain is comprised of a light chain variable region (VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells). The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from the amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of each heavy/light chain pair (VH/VL) typically form an antibody's antigen-binding site. The term “antibody” is not limited by any particular method of producing the antibody. For example, it includes monoclonal antibodies, recombinant antibodies, and polyclonal antibodies.

The term “human antibody” refers to an antibody consisting of amino acid sequences of human immunoglobulin sequences only. A human antibody may contain murine carbohydrate chains if produced in a mouse, in a mouse cell or in a hybridoma derived from a mouse cell. Human antibodies may be prepared in a variety of ways known in the art.

The term “humanized antibody” refers to an antibody that contains some or all of the CDRs from a non-human animal antibody, while the framework and constant regions of the antibody contain amino acid residues derived from human antibody sequences. Humanized antibodies are typically produced by grafting CDRs from a mouse antibody into human framework sequences followed by back substitution of certain human framework residues for the corresponding mouse residues from the source antibody. The term “humanized antibody” also refers to an antibody of non-human origin in which, typically in one or more variable regions, one or more epitopes have been removed that have a high propensity of constituting a human T-cell and/or B-cell epitope, for purposes of reducing immunogenicity. The amino acid sequence of the epitope can be removed in full or in part. However, typically the amino acid sequence is altered by substituting one or more of the amino acids constituting the epitope for one or more other amino acids, thereby changing the amino acid sequence into a sequence that does not constitute a human T-cell and/or B-cell epitope. The amino acids are substituted by amino acids that are present at the corresponding position(s) in a corresponding human variable heavy or variable light chain as the case may be.

The term “pharmaceutically acceptable carrier” means solvents, carrier agents, diluting agents and the like which are usually used in the administration of pharmaceutical compounds.

CD47 is overexpressed in various cancer cells, in virus-infected cells, and in fibrotic cells, offering a promising target for the treatment of various cancers, viral infections, and fibrotic diseases. By blocking CD47 signaling in these cells, and even the expression of CD47 itself, these cells' evasion of the immune system can be suppressed, allowing for their elimination and the subsequent recovery of a patient from disease.

For example, intravenous injection of a variant SIRPα (“vSIRPα”), having high affinity for CD47 and conjugated to a probe, has been found to be taken up by tumorigenic cells of mice. In addition, vSIRPα, conjugated to a siRNA targeting CD47, has been shown to enhance phagocytosis of CT26.CL25 cancer cells in culture (Ko et al. Control Release 323:376-386 (2020) and U.S. Publication No. 2021/0015931, each of which is hereby incorporated by reference herein in its entirety).

We have unexpectedly found that CD47 present on the cell surface of red blood cells (“RBC”) and on the cell surface of target cells, such as cancer cells, virus-infected cells, and fibrotic cells, can be utilized to deliver various APIs to said target cells via a novel mechanism.

Here, we describe novel therapeutic compounds for RBC-mediated delivery of an API to a target cell expressing CD47 in a mammalian subject. The therapeutic compound is a conjugate comprising a CD47-binding protein conjugated to an API. The CD47-binding protein of the conjugate binds the conjugate to CD47 present on the surface of the subject's red blood cells, thereby allowing transport of the conjugate through the subject's circulatory system to the target cell. The conjugate is transferred from the RBC to CD47 present on the surface of the target cell. Upon binding of the conjugate to CD47 of the target cell, the ability of the target cell to evade attack by the subject's immune system is reduced. Furthermore, upon binding of the conjugate to CD47 of the target cell, the conjugate-CD47 complex is internalized via endocytosis, further reducing the ability of the target cell to evade attack by the subject's immune system, and delivering the API into the target cell. Surprisingly, when the conjugate is bound to CD47 on the surface of the RBC, the conjugate-CD47 is not internalized by the RBC. In some embodiments, the mammalian subject is a human.

The CD47-binding protein may be conjugated to the API by a linker. A linker connects a CD47-binding protein to an API. The linker may be a cleavable linker that is cleaved upon internalization of conjugate by the target cell, thereby releasing the API from the CD47-binding protein

In some embodiments, the target cell is a cancer cell and the therapeutic compound may be used to treat a cancer in a mammalian subject.

The cancer cell may be in a tumor attributable to a cancer selected from the group consisting of brain tumor, spinal cord tumor, retinoblastoma, oral cancer, nasal cavity cancer, paranasal sinus cancer, pharyngeal cancer, laryngeal cancer, neck cancer, head and neck cancer, melanoma, skin cancer, breast cancer, thyroid cancer, malignant adrenal tumor, endocrine cancer, lung cancer, pleural tumor, respiratory tract cancer, esophageal cancer, stomach cancer, small intestine cancer, colon cancer, anal cancer, liver cancer, biliary tract cancer, pancreatic cancer, kidney cancer, bladder cancer, prostate cancer, testicular cancer, penile cancer, cervical cancer, endometrial cancer, choriocarcinoma, ovarian cancer, blood cancer including acute/chronic leukemia, malignant lymphoma and multiple myeloma, bone tumor, soft tissue tumor, childhood leukemia, and childhood cancer.

The cancer cell may attributable to a cancer selected from the group consisting of ovarian serous cystadenocarcinoma, lung adenocarcinoma, cervical and endocervical cancer, head and neck squamous cell carcinoma, thyroid carcinoma, uterine corpus endometrioid carcinoma, prostate adenocarcinoma, mesothelioma, diffuse large B-cell lymphoma, acute leukemia, lung squamous cell carcinoma, acute lymphoblastic leukemia, esophageal carcinoma, myxofibrosarcoma, pancreatic adenocarcinoma, rectum adenocarcinoma, colon adenocarcinoma, acute megakaryoblastic leukemia, breast invasive carcinoma, stomach adenocarcinoma, bladder urothelial carcinoma, cholangiocarcinoma, leukemia, thymic carcinoma, leiomyosarcoma, thymoma, undifferentiated pleomorphic sarcoma, uterine carcinosarcoma, acute myeloid leukemia, glioblastoma multiforme, sarcoma, skin cutaneous melanoma, kidney clear cell carcinoma, dedifferentiated liposarcoma, lymphoma, retinoblastoma, neuroblastoma, osteosarcoma, juvenile myelomonocytic leukemia, gastrointestinal stromal tumor, dysembryoplatic neuroepithelial tumor, adrenocortical cancer, acute leukemia of ambiguous lineage, pheochromocytoma and paraganglioma, glioma, testicular germ cell tumor, supratentorial embryonal tumor NOS, neurofibroma, kidney papillary cell carcinoma, hepatocellular carcinoma, kidney chromophobe, malignant peripheral nerve sheath tumor, ependymoma, adrenocortical carcinoma, nasopharyngeal carcinoma, spindle cells/sclerosing rhabdomyosarcoma, melanoma, choroid plexus carcinoma, undifferentiated spindle cell carcinoma, myoepithelial carcinoma, alveolar rhabdomyosarcoma, rhabdomyosarcoma, atypical teratoid/rhabdoid tumor, desmoplastic small round cell tumor, fibromatosis, synovial sarcoma, wilms tumor, myofibromytosis, fibrolamellar hepatocellular carcinoma, undifferentiated sarcoma NOS, embryonal rhabdomyosarcoma, uveal melanoma, Ewing sarcoma, hepatoblastoma, infantile fibrosarcoma, INI-deficient soft tissue sarcoma NOA, undifferentiated hepatic sarcoma, and medulloblastoma. See Gupta et al. Cancer Drug Resist 3:550-62 (2020), which is hereby incorporated by reference herein in its entirety.

In other embodiments, the target cell is a virus-infected cell and the therapeutic compound may be used to treat a viral infection in a mammalian subject. The virus-infected cell may be infected with the SARS-CoV-2 virus.

In some embodiments, the target cell is a fibrotic cell and the therapeutic compound may be used to treat a fibrotic disease in a mammalian subject. The fibrotic cell may be a fibrotic fibroblast. In some embodiments, the fibrotic disease is cystic fibrosis.

CD47-Binding Proteins

Suitable CD47-binding proteins for the conjugates described herein include wild type (“wt”) SIRPα (SEQ ID NO:1), variant SIRPα (“vSIRPα”) (SEQ ID NO:3), wt TSP-1 (SEQ ID NO:7), wt SIRPγ (SEQ ID NO:4), variant SIRPγ-1 (“vSIRPγ-1”) (SEQ ID NO:5), variant SIRPγ-2 (“vSIRPγ-2”) (SEQ ID NO:6), and homologs of any of the foregoing. ALX148 (SEQ ID NO: 962), TTI-661 (SEQ ID NO: 963), TTI-662 (SEQ ID NO: 964), and homologs thereof are also suitable CD47-binding proteins for the conjugates described herein. ALX148 is a SIRPα D1 variant fused to an Fc domain monomer. See, e.g., U.S. Pat. No. 10,696,730, which is hereby incorporated by reference herein in its entirety. TTI-661 is an IgV domain of human SIRPα variant 2 fused to a constant region of human IgG1 antibody, and TTI-662 is an IgV domain of human SIRPα variant 2 fused to a constant region of human IgG4 antibody. See, e.g., U.S. Pat. No. 9,969,789, which is hereby incorporated by reference herein in its entirety.

Other suitable CD47-binding proteins include anti-CD47 antibodies.

In some embodiments, the CD47-binding protein is an anti-CD47 antibody, such as B6H12, 5F9, 8B6, C3, and Hu5F9-G4, described in U.S. Pat. Nos. 9,017,675 and 9,623,079, each of which is hereby incorporated by reference herein in its entirety.

In some embodiments, the anti-CD47 antibody includes a heavy chain variable region comprising SEQ ID NO: 930 and a light chain variable region comprising SEQ ID NO: 931. In some embodiments, the anti-CD47 antibody includes a heavy chain variable region comprising SEQ ID NO: 938 and a light chain variable region comprising SEQ ID NO: 939. In some embodiments, the anti-CD47 antibody includes a heavy chain variable region comprising SEQ ID NO: 946 and a light chain variable region comprising SEQ ID NO: 947. In some embodiments, the anti-CD47 antibody includes a heavy chain variable region comprising SEQ ID NO: 954 and a light chain variable region comprising SEQ ID NO: 955.

In some embodiments, the anti-CD47 antibody comprises a heavy chain variable region including complementarity determining regions CDR1, CDR2, and CDR3 comprising SEQ ID NO: 932, SEQ ID NO: 933, and SEQ ID NO: 934, respectively, and a light chain variable region including complementarity determining regions CDR1, CDR2, and CDR3 comprising SEQ ID NO: 935, SEQ ID NO: 936, and SEQ ID NO: 937, respectively.

In some embodiments, the anti-CD47 antibody comprises a heavy chain variable region including complementarity determining regions CDR1, CDR2, and CDR3 comprising SEQ ID NO: 940, SEQ ID NO: 941, and SEQ ID NO: 942, respectively, and a light chain variable region including complementarity determining regions CDR1, CDR2, and CDR3 comprising SEQ ID NO: 943, SEQ ID NO: 944, and SEQ ID NO: 945, respectively.

In some embodiments, the anti-CD47 antibody comprises a heavy chain variable region including complementarity determining regions CDR1, CDR2, and CDR3 comprising SEQ ID NO: 948, SEQ ID NO: 949, and SEQ ID NO: 950, respectively, and a light chain variable region including complementarity determining regions CDR1, CDR2, and CDR3 comprising SEQ ID NO: 951, SEQ ID NO: 952, and SEQ ID NO: 953, respectively.

In some embodiments, the anti-CD47 antibody comprises a heavy chain variable region including complementarity determining regions CDR1, CDR2, and CDR3 comprising SEQ ID NO: 956, SEQ ID NO: 957, and SEQ ID NO: 958, respectively, and a light chain variable region including complementarity determining regions CDR1, CDR2, and CDR3 comprising SEQ ID NO: 959, SEQ ID NO: 960, and SEQ ID NO: 961, respectively.

In some embodiments, the anti-CD47 antibody is a human antibody. In some embodiments, the anti-CD47 antibody is a humanized antibody.

Each of these CD47-binding proteins binds to CD47 present on the surface of a target cell.

In some embodiments, a CD47-binding protein is conjugated to an API by a bond selected from the group consisting of a covalent bond, a hydrogen bond, an ionic bond, a van der Waals interaction, and combinations thereof. Examples of linkers that covalently bond a CD47-binding protein to an API are described below.

Linkers

Suitable linkers include, but are not limited to, cleavable linkers such as hydrazone linkers, imine linkers, oxime linkers, carbonate linkers, acetal linkers, orthoester linkers, silyl ether linkers, disulfide linkers, trioxolane linkers, beta-glucuronide linkers, beta-galactoside linkers, pyrophosphate linkers, phosphoramidate linkers, arylsulfate linkers, heptamethine cyanine linkers, nitrobenzyl linkers, aryl boronic acid linkers, boronate linkers, thioether linkers, maleimidocaproyl-containging linkers, enzyme-cleavable peptide linkers, and para-amino benzyl carbamate-containing linkers, as well as non-cleavable linkers such as polyethylene glycol.

The CD47-binding protein-API conjugates described herein may be made by joining the CD47-binding protein to the API via a linker using coupling reactions such as bis(vinylsulfonyl)piperazine-disulfide coupling, N-methyl-N-phenylvinylsulfonamide-cysteine coupling, platinum (II) compound-histidine coupling, and tetrazine-trans-cyclooctene coupling. Suitable linkers and coupling reactions are known to those of skill in art. See, e.g., Su et al. Acta Pharmaceutica Sinica B (2021), ISSN 2211-3835; Pan et al. Med Res Rev. 40:2682-2713 (2020); Khongorzulet al. Mol Cancer Res 18:3-19 (2020); Bargh et al. Chem Soc Rev 48:4361-4374 (2019); and Smith et al. Pharm Res 32:3526-3540 (2015), each of which is hereby incorporated by reference herein in its entirety.

Various enzyme-cleavable peptide linkers, such as those described below, can be used to couple a CD47-binding protein to an API to form a CD47-binding protein-API conjugate, in accordance with embodiments of the invention. These linkers comprise amino acid residues and are cleaved by specific enzymes within a cell, such as lysosomal degradative enzymes. See, e.g., Kong et al. J Biol Chem 290:7160-7168 (2015); Poreba FEBS J 287:1936-1969 (2020); and Singh et al., Current Medicinal Chemistry 15(18) (2008), each of which is hereby incorporated by reference herein in its entirety. Exemplary peptide linkers suitable for use in accordance with embodiments of the invention are described below.

For example, di-peptide linkers are comprised of two amino acid residues that serve as a recognition motif for cleavage by the enzyme cathepsin B, which cleaves the amide bond after the second amino acid residue between the carbonyl and amine. Di-peptide linkers cleaved by cathepsin B include Phe-Arg, Phe-Cit, Phe-Lys, Ala-Arg, Ala-Cit, Val-Ala, Val-Arg, Val-Lys, Val-Cit, and Arg-Arg. Cathepsin B similarly recognizes and cleaves the tetra-peptide linkers Gly-Phe-Leu-Gly and Ala-Leu-Ala-Leu after the fourth amino acid residue.

In addition, the tri-peptide linker Ala-Ala-Asn is cleaved by the enzyme legumain after the last amino acid residue. The tetra-peptide linkers Lys-Ala-Gly-Gly, Leu-Arg-Gly-Gly, and Arg-Lys-Arg-Arg are cleaved by the papain-like protease enzyme.

Peptide linkers Arg-Arg-X, Ala-Leu-X, Gly-Leu-Phe-Gly-X, Gly-Phe-Leu-Gly-X, and Ala-Leu-Ala-Leu-X, where X is any amino acid, are cleaved by the enzymes cathepsin B, H, and L. Cathepsin B, H, and L are responsible for lysosomal degradation of proteins.

Peptide linkers Phe-Ala-Ala-Phe(NO₂)-Phe-Val-Leu-OM4P-X and Bz-Arg-Gly-Phe-Phe-Pro-4mβNA, where X is any amino acid, are cleaved by the enzyme cathepsin D.

Serum plasminogen activator is produced in many tumor cells. Plasminogen is converted to plasmin, thus producing a high-level of plasmin in the tumor cells. This plasmin is degraded rapidly in the plasma and hence tissues remote to the tumor are not exposed to plasmin. Plasmin is responsible for the fibrinolysis and degradation of blood plasma proteins and cleaves the peptide linkers D-Val-Leu-Lys-X, D-Ala-Phe-Lys-X, and D-Ala-Trp-Lys-X, where X is any amino acid.

Tissue plasminogen activator (tPA) and urokinase (uPA) are responsible for activation of plasmin formation and can each cleave the peptide linker Gly-Gly-Gly-Arg-Arg-Arg-Val-X, where X is any amino acid.

Prostate-specific antigen is responsible for liquefaction of semen and cleaves the peptide linker morpholinocarbonyl-His-Ser-Ser-Lys-Leu-Gln-Leu-X, where X is any amino acid.

Matrix metalloproteases (MMP-2 and MM-9) are responsible for degradation of extracellular matrix and collagens and cleave the peptide linkers Ac-Pro-Leu-Gln-Leu-X and Gly-Pro-Leu-Gly-Ile-Ala-Gly-Gln-X, where X is any amino acid.

APIs

In accordance with some embodiments, the API may be a small molecule. For example, small molecule APIs useful in the treatment of cancer include, but are not limited to, methotrexate; doxorubicin; vinca alkaloids; camptothecin analogues; microtubule-disrupting agents such as auristatins (e.g., MMAE and MMAF) and maytansinoids (e.g., DM1 and DM4); and DNA-damaging agents such as DNA topoisomerase I inhibitors (e.g., SN-38 and exatecan), double-strand break agents (e.g., calicheamicin), cross-linkers (e.g., pyrrolobenzodiazepine dimer-PBD), and alkylators (e.g., duocarmycin and indolinobenzodiazepine dimer-IGN). See, e.g., Khongorzulet al. Mol Cancer Res 18:3-19 (2020); Salomon et al. Mol Pharm 16(12):4817-4825 (2019); and Drago et al. Nat Rev Clin Oncol 18, 327-344 (2021), each of which is hereby incorporated by reference herein in its entirety.

In accordance with other embodiments, the API may be small-interfering RNA (“siRNA”). siRNA is a double-stranded RNA molecule that can reduce the expression of a specific gene by causing the degradation of the gene's mRNA transcript(s), which shares partial complementarity with a strand of the double-stranded siRNA molecule. The process of reducing the expression of a gene using siRNA is referred to as RNA interference (“RNAi”). See U.S. Pat. Nos. 7,056,704, 7,078,196, 8,372,968 each of which is hereby incorporated by reference herein its entirety.

Several genes have been implicated in promoting cancer progression and cancer cell proliferation through various mechanisms. These genes, and their mRNA transcripts are listed in Table 1. By reducing the expression of one or more genes from Table 1, cancer progression and cancer cell proliferation may be inhibited. Thus, the transcripts of the genes listed in Table 1 represent key targets for the treatment of cancer using siRNA-mediated RNAi.

In some embodiments, an API useful for the treatment of cancer in a mammal is siRNA, the siRNA being a double-stranded RNA molecule including an antisense RNA strand and a sense RNA strand, wherein: (a) the antisense RNA strand is 19-29 nucleotides in length and is complementary to contiguous nucleotides in a target mammalian mRNA sequence, the mRNA sequence being selected from the group consisting of SEQ ID NO: 8-747 and 771-824, (b) the sense RNA strand is 19-29 nucleotides in length and is complementary to 14-29 nucleotides from the antisense RNA strand, and (c) the double stranded RNA molecule has a double stranded region of 14-29 nucleotides in length and a 3′ overhang region of 0-5 nucleotides in length.

In some embodiments, an API useful for the treatment of cancer in a mammal is siRNA, the siRNA being a double-stranded RNA molecule including an antisense RNA strand and a sense RNA strand, wherein: (a) the antisense RNA strand is 19-29 nucleotides in length and is complementary to contiguous nucleotides in a target mammalian mRNA sequence, the mRNA sequence being selected from the group consisting of SEQ ID NO: 22-747 and 771-824, (b) the sense RNA strand is 19-29 nucleotides in length and is complementary to 14-29 nucleotides from the antisense RNA strand, and (c) the double stranded RNA molecule has a double stranded region of 14-29 nucleotides in length and a 3′ overhang region of 0-5 nucleotides in length.

In some embodiments, an API useful for the treatment of cancer in a mammal is siRNA, the siRNA being a double-stranded RNA molecule including an antisense RNA strand and a sense RNA strand, wherein: (a) the antisense RNA strand is 19-29 nucleotides in length and is complementary to contiguous nucleotides in a target mammalian mRNA sequence, the mRNA sequence selected from the group consisting of SEQ ID NO: 22-37, (b) the sense RNA strand is 19-29 nucleotides in length and is complementary to 14-29 nucleotides from the antisense RNA strand, and (c) the double stranded RNA molecule has a double stranded region of 14-29 nucleotides in length and a 3′ overhang region of 0-5 nucleotides in length.

In some embodiments, an API useful for the treatment of cancer in a mammal is siRNA, the siRNA being a double-stranded RNA molecule including an antisense RNA strand and a sense RNA strand, wherein: (a) the antisense RNA strand is 19-29 nucleotides in length and is complementary to contiguous nucleotides in a target mammalian mRNA sequence, the mRNA sequence selected from the group consisting of SEQ ID NO: 38-39, (b) the sense RNA strand is 19-29 nucleotides in length and is complementary to 14-29 nucleotides from the antisense RNA strand, and (c) the double stranded RNA molecule has a double stranded region of 14-29 nucleotides in length and a 3′ overhang region of 0-5 nucleotides in length.

In some embodiments, an API useful for the treatment of cancer in a mammal is siRNA, the siRNA being a double-stranded RNA molecule including an antisense RNA strand and a sense RNA strand, wherein: (a) the antisense RNA strand is 19-29 nucleotides in length and is complementary to contiguous nucleotides in a target mammalian mRNA sequence, the mRNA sequence selected from the group consisting of SEQ ID NO: 40-43, (b) the sense RNA strand is 19-29 nucleotides in length and is complementary to 14-29 nucleotides from the antisense RNA strand, and (c) the double stranded RNA molecule has a double stranded region of 14-29 nucleotides in length and a 3′ overhang region of 0-5 nucleotides in length.

In some embodiments, an API useful for the treatment of cancer in a mammal is siRNA, the siRNA being a double-stranded RNA molecule including an antisense RNA strand and a sense RNA strand, wherein: (a) the antisense RNA strand is 19-29 nucleotides in length and is complementary to contiguous nucleotides in a target mammalian mRNA sequence, the mRNA sequence selected from the group consisting of SEQ ID NO: 44-51, (b) the sense RNA strand is 19-29 nucleotides in length and is complementary to 14-29 nucleotides from the antisense RNA strand, and (c) the double stranded RNA molecule has a double stranded region of 14-29 nucleotides in length and a 3′ overhang region of 0-5 nucleotides in length.

TABLE 1 siRNA and shRNA Cancer (Cancer Cell) Target Transcripts (mRNA) GenBank GenBank cDNA SEQ ID Gene Gene ID RefSeq Accession NO CD47 961 NM_001777.4 8 NM_198793.3 9 NM_001382306.1 10 XM_005247909.2 11 XM_017007536.1 12 XR_241521.2 13 XR_241522.2 14 XR_924218.2 15 XR_924219.2 16 XR_924220.2 17 XR_001740374.2 18 XR_001740375.2 19 XR_002959610.1 20 XR_002959611.1 21 KRAS 3845 NM_004985.5 22 NM_033360.4 23 NM_001369786.1 24 NM_001369787.1 25 NM_004985.5 w/G→T  26 substitution at position 34 NM_033360.4 w/G→T  27 substitution at position 34 NM_001369786.1 w/G→T  28 substitution at position 34 NM_001369787.1 w/G→T  29 substitution at position 34 NM_004985.5 w/G→A  30 substitution at position 35 NM_033360.4 w/G→A  31 substitution at position 35 NM_001369786.1 w/G→A  32 substitution at position 35 NM_001369787.1 w/G→A  33 substitution at position 35 NM_004985.5 w/G→T  34 substitution at position 35 NM_033360.4 w/G→T  35 substitution at position 35 NM_001369786.1 w/G→T  36 substitution at position 35 NM_001369787.1 w/G→T  37 substitution at position 35 C-MYC 4609 NM_002467.6 38 NM_001354870.1 39 CD274 29126 NM_014143.4 40 NM_001267706.2 41 NM_001314029.2 42 NR_052005.2 43 CD24 100133941 NM_001359084.1 44 NM_013230.3 45 NM_001291737.1 46 NM_001291738.1 47 NM_001291739.1 48 NR_117089.1 49 NR_117090.1 50 XM_024446293.1 51 BIRC5 332 NM_001168.3 52 NM_001012270.2 53 NM_001012271.2 54 XR_243654.5 55 XR_934452.3 56 PKN3 29941 NM_013355.5 57 NM_001317926.2 58 XM_005251946.3 59 XM+006717080.3 60 XM_017014649.2 61 XM_017014650.1 62 PLK1 5347 NM_005030.6 63 FGF1 2246 NM_000800.5 64 NM_033136.4 65 NM_033137.4 66 NM_001144892.3 67 NM_001144934.2 68 NM_001144935.2 69 NM_001257205.1 70 NM_001257206.2 71 NM_001257207.2 72 NM_001257208.2 73 NM_001257209.1 74 NM_001257210.2 75 NM_001257211.2 76 NM_001257212.2 77 NM_001354951.2 78 NM_001354952.2 79 NM_001354953.2 80 NM_001354954.2 81 NM_001354955.2 82 NM_001354956.2 83 NM_001354957.2 84 NM_001354958.2 85 NM_001354959.2 86 NM_001354961.2 87 NM_001354962.2 88 NM_001354963.2 89 NM_001354964.2 90 FGF2 2247 NM_001361665.2 91 NM_002006.5 92 FGF5 2250 NM_004464.4 93 NM_033143.2 94 NM_001291812.2 95 EGFR 1956 NM_005228.5 96 NM_201282.2 97 NM_201283.2 98 NM_201284.2 99 NM_001346897.2 100 NM_001346898.2 101 NM_001346899.2 102 NM_001346900.2 103 NM_001346941.2 104 VEGFA 7422 NM_003376.6 105 NM_001025366.3 106 NM_001025367.3 107 NM_001025368.3 108 NM_001025369.3 109 NM_001025370.3 110 NM_001033756.3 111 NM_001171622.2 112 NM_001171623.2 113 NM_001171624.2 114 NM_001171625.2 115 NM_001171626.2 116 NM_001171627.2 117 NM_001171628.2 118 NM_001171629.2 119 NM_001171630.2 120 NM_001204384.2 121 NM_001204385.2 122 NM_001287044.2 123 NM_001317010.1 124 VEGFB 7423 NM_003377.5 125 NM_001243733.2 126 KDR 3791 NM_002253.4 127 ERBB2 2064 NM_004448.4 128 NM_001005862.3 129 NM_001289936.2 130 NM_001289937.2 131 NM_001289938.2 132 NM_001382782.1 133 NM_001382783.1 134 NM_001382784.1 135 NM_001382785.1 136 NM_001382786.1 137 NM_001382787.1 138 NM_001382788.1 139 NM_001382789.1 140 NM_001382790.1 141 NM_001382791.1 142 NM_001382792.1 143 NM_001382793.1 144 NM_001382794.1 145 NM_001382795.1 146 NM_001382796.1 147 NM_001382797.1 148 NM_001382798.1 149 NM_001382799.1 150 NM_001382800.1 151 NM_001382801.1 152 NM_001382802.1 153 NM_001382803.1 154 NM_001382804.1 155 NM_001382805.1 156 NM_001382806.1 157 NR_110535.2 158 XM_024450641.1 159 XM_024450642.1 160 XM_024450643.1 161 EPHA2 1969 NM_004431.5 162 NM_001329090.2 163 XM_017000537.1 164 RRM2 6241 NM_001034.4 165 NM_001165931.1 166 NR_164157.1 167 PSMB9 5698 NM_002800.5 168 PSMB8 5696 NM_148919.4 169 NM_004159.5 170 MCL1 4170 NM_021960.5 171 NM_182763.3 172 NM_001197320.2 173 CBLB 868 NM_170662.5 174 NM_001321786.1 175 NM_001321788.2 176 NM_001321789.1 177 NM_001321790.2 178 NM_001321791.2 179 NM_001321793.2 180 NM_001321794.2 181 NM_001321795.2 182 NM_001321796.2 183 NM_001321797.2 184 NM_001321798.2 185 NM_001321799.2 186 NM_001321806.2 187 NM_001321807.2 188 NM_001321808.2 189 NM_001321811.2 190 NM_001321813.1 191 NM_001321816.2 192 NM_001321820.2 193 NM_001321822.2 194 NR_135806.2 195 NR_135807.2 196 NR_135808.2 197 NR_135809.2 198 NR_135810.2 199 NR_135811.2 200 NR_135812.1 201 XM_011513257.1 202 XM_011513259.3 203 XM_017007395.1 204 XM_017007397.1 205 XM_017007396.1 206 XM_017007399.1 207 XM_017007398.1 208 XM_017007400.1 209 XR_001740338.1 210 XR_001740339.1 211 RHOA 387 NM_001664.4 212 NM_001313941.2 213 NM_001313943.2 214 NM_001313944.2 215 NM_001313945.2 216 NM_001313946.2 217 NM_001313947.2 218 FLI1 2313 NM_002017.5 219 NM_001167681.3 220 NM_001271010.2 221 NM_001271012.2 222 XM_011542701.2 223 XM_011542702.1 224 XM_017017405.1 225 XM_017017406.1 226 EWSR1 2130 NM_005243.4 227 NM_013986.4 228 NM_001163285.2 229 NM_001163286.2 230 NM_001163287.2 231 XM_005261389.4 232 XM_005261390.4 233 XM_011529995.3 234 XM_011529996.3 235 XM_011529997.2 236 XM_011529998.2 237 XM_011529999.3 238 XM_011530000.2 239 XM_011530001.2 240 XM_011530002.3 241 XM_017028644.2 242 XM_017028645.2 243 XM_017028646.2 244 XM_017028647.2 245 XM_017028649.2 246 XM_017028648.2 247 XM_017028650.2 248 XM_017028651.2 249 XM_017028652.2 250 XM_017028653.2 251 XM_017028654.1 252 XM_017028655.1 253 XM_017028656.2 254 XM_017028657.2 255 XM_017028659.1 256 XM_017028658.1 257 XM_017028660.2 258 XM_017028661.2 259 XM_017028662.2 260 XM_017028663.1 261 XM_017028664.2 262 XM_017028665.2 263 XM_017028666.2 264 XM_024452180.1 265 XM_024452181.1 266 XR_002958676.1 267 STAT3 6774 NM_139276.3 268 NM_003150.4 269 NM_213662.2 270 NM_001369512.1 271 NM_001369513.1 272 NM_001369514.1 273 NM_001369516.1 274 NM_001369517.1 275 NM_001369518.1 276 NM_001369519.1 277 NM_001369520.1 278 NM_001384984.1 279 NM_001384985.1 280 NM_001384986.1 281 NM_001384987.1 282 NM_001384988.1 283 NM_001384989.1 284 NM_001384990.1 285 NM_001384991.1 286 NM_001384992.1 287 NM_001384993.1 288 XM_017024973.2 289 XM_024450896.1 290 TWIST1 7291 NM_000474.4 291 NR_149001.2 292 FOLH1 2346 NM_004476.3 293 NM_001014986.3 294 NM_001193471.3 295 NM_001193472.3 296 NM_001193473.3 297 NM_001351236.2 298 XM_011519958.3 299 XM_017017432.1 300 XM_017017433.2 301 XM_017017434.1 302 XM_017017435.2 303 XM_017017444.2 304 XM_017017445.1 305 XM_017017446.1 306 XM_017017447.1 307 XM_017017448.1 308 XM_017017449.2 309 XM_017017450.2 310 XM_017017451.2 311 XM_024448411.1 312 XR_001747818.1 313 XR_001747819.1 314 HIF1A 3091 NM_001530.4 315 NM_181054.3 316 NM_001243084.2 317 SERPINH1 871 NM_001235.5 318 NM_001207014.3 319 XM_011545327.1 320 XM_024448756.1 321 PTK2 5747 NM_001387646.1 322 NM_005607.5 323 NM_153831.4 324 NM_001199649.2 325 NM_001316342.2 326 NM_001352694.2 327 NM_001352695.2 328 NM_001352696.2 329 NM_001352697.2 330 NM_001352698.2 331 NM_001352699.2 332 NM_001352700.2 333 NM_001352701.2 334 XM_017013654.2 335 NM_001352703.2 336 NM_001352704.2 337 NM 001352705.2 338 NM_001352706.2 339 NM 001352707.2 340 NM_001352708.2 341 NM_001352709.2 342 NM_001352710.2 343 NM_001352711.2 344 NM_001352712.2 345 NM_001352713.2 346 NM_001352714.2 347 NM 001352715.2 348 NM 001352716.2 349 NM_001352717.2 350 NM_001352718.2 351 NM_001352719.2 352 NM_001352720.2 353 NM_001352721.2 354 NM_001352722.2 355 NM 001352723.2 356 NM_001352724.2 357 NM 001352725.2 358 NM_001352726.2 359 NM_001352727.2 360 NM 001352728.2 361 NM 001352729.2 362 NM_001352730.2 363 NM 001352731.2 364 NM_001352732.2 365 NM_001352733.2 366 NM_001352734.2 367 NM_001352735.2 368 NM_001352736.2 369 NM_001352737.2 370 NM 001352738.2 371 NM_001352739.2 372 NM_001352740.2 373 NM_001352741.2 374 NM 001352742.2 375 NM_001352743.2 376 NM 001352744.2 377 NM_001352745.2 378 NM_001352746.2 379 NM_001352747.2 380 NM 001352748.2 381 NM_001352749.2 382 NM_001352750.2 383 NM_001352751.2 384 NM 001352752.2 385 NM 001387584.1 386 NM_001387585.1 387 NM_001387586.1 388 NM_001387587.1 389 NM_001387588.1 390 NM_001387589.1 391 NM_001387590.1 392 NM 001387591.1 393 NM_001387592.1 394 NM 001387603.1 395 NM_001387604.1 396 NM_001387605.1 397 NM_001387606.1 398 NM 001387607.1 399 NM_001387608.1 400 NM_001387609.1 401 NM 001387610.1 402 NM 001387611.1 403 NM 001387612.1 404 NM_001387613.1 405 NM_001387614.1 406 NM_001387615.1 407 NM_001387616.1 408 NM_001387617.1 409 NM_001387618.1 410 NM_001387619.1 411 NM 001387620.1 412 NM 001387621.1 413 NM_001387622.1 414 NM_001387623.1 415 NM_001387624.1 416 NM_001387625.1 417 NM_001387627.1 418 NM_001387628.1 419 NM_001387629.1 420 NM_001387630.1 421 NM_001387631.1 422 NM 001387632.1 423 NM_001387633.1 424 NM_001387634.1 425 NM_001387635.1 426 NM_001387636.1 427 NM_001387637.1 428 NM_001387638.1 429 NM_001387639.1 430 NM_001387640.1 431 NM_001387641.1 432 NM_001387642.1 433 NM_001387643.1 434 NM_001387644.1 435 NM_001387645.1 436 NM_001387647.1 437 NM_001387648.1 438 NM_001387649.1 439 NM_001387650.1 440 NM_001387651.1 441 NM_001387652.1 442 NM_001387653.1 443 NM_001387654.1 444 NM_001387655.1 445 NM_001387656.1 446 NM_001387657.1 447 NM_001387658.1 448 NM_001387659.1 449 NM_001387660.1 450 NM_001387661.1 451 NM_001387662.1 452 NR 148036.2 453 NR 148037.2 454 NR 148038.2 455 NR 148039.2 456 NR 170670.1 457 NR 170671.1 458 NR 170672.1 459 NR_170673.1 460 XM_017013654.2 461 XM_017013656.2 462 XM_017013666.1 463 XM_017013669.2 464 XM_017013684.2 465 XM_017013688.2 466 XM_024447199.1 467 XM_024447200.1 468 XM_024447202.1 469 XM_024447201.1 470 XM_024447204.1 471 XM_024447203.1 472 XM_024447206.1 473 XM_024447205.1 474 XM_024447208.1 475 XM_024447207.1 476 XM_024447210.1 477 XM_024447209.1 478 XM_024447211.1 479 CEACAM6 4680 NM_002483.7 480 XM_011526990.2 481 CXCR4 7852 NM_003467.3 482 NM_001008540.2 483 NM_001348056.2 484 NM_001348059.2 485 NM_001348060.2 486 CTNNB1 1499 NM_001904.4 487 NM_001098209.2 488 NM_001098210.2 489 NM_001330729.2 490 XM_006712983.2 491 XM_006712985.1 492 XM_017005738.1 493 XM_024453356.1 494 XM_024453357.1 495 XM_024453358.1 496 XM_024453359.1 497 XM_024453360.1 498 BCL2 596 NM_000633.3 499 NM_000657.3 500 XM_011526135.3 501 XM_017025917.2 502 XR_935248.3 503 BCL2L1 598 NM_138578.3 504 NM_001191.4 505 NM_001317919.2 506 NM_001317920.2 507 NM_001317921.2 508 NM_001322239.2 509 NM_001322240.2 510 NM_001322242.2 511 NR_134257.1 512 XM_011528964.2 513 XM_017027993.1 514 XR_936599.3 515 XR_001754364.2 516 SST 6750 NM_001048.4 517 RAF1 5894 NM_001354689.3 518 NM_002880.4 519 NM_001354690.3 520 NM_001354691.3 521 NM_001354692.3 522 NM_001354693.3 523 NM_001354694.3 524 NM_001354695.3 525 NR_148940.3 526 NR_148941.3 527 NR_148942.3 528 XM_011533974.3 529 XM_017006966.1 530 XR_001740227.1 531 SKP2 6502 NM_005983.4 532 NM_032637.4 533 NM_001243120.2 534 XM_011514082.3 535 XM_011514083.3 536 XM_017009753.1 537 XR_001742203.2 538 PLAUR 5329 NM_002659.4 539 NM_001005376.3 540 NM_001005377.3 541 NM_001301037.2 542 XM_005258990.5 543 XM_011527027.2 544 XM_011527028.3 545 XM_011527029.2 546 XM_011527030.2 547 XM_011527031.3 548 XM_017026872.2 549 XM_017026873.1 550 MDM2 4193 NM_002392.6 551 NM_001145337.3 552 NM_001145339.2 553 NM_001145340.3 554 NM_001278462.2 555 NM_001367990.1 556 XM_006719399.4 557 XM_006719400.4 558 RAD51 5888 NM_002875.5 559 NM_133487.4 560 NM_001164269.2 561 NM_001164270.2 562 XM_006720626.3 563 XM_011521857.2 564 XM_011521858.2 565 XM_011521859.2 566 XM_011521860.2 567 XM_011521861.2 568 XM_011521862.3 569 EZH2 2146 NM_004456.5 570 NM_152998.3 571 NM_001203247.2 572 NM_001203248.2 573 NM_001203249.2 574 XM_005249962.4 575 XM_005249963.4 576 XM_005249964.4 577 XM_011515883.2 578 XM_011515884.2 579 XM_011515885.2 580 XM_011515886.2 581 XM_011515887.3 582 XM_011515888.2 583 XM_011515889.2 584 XM_011515890.2 585 XM_011515891.3 586 XM_011515892.2 587 XM_011515893.2 588 XM_011515894.2 589 XM_011515895.2 590 XM_011515896.2 591 XM_011515897.2 592 XM_011515898.2 593 XM_011515899.3 594 XM_011515901.3 595 XM_017011817.2 596 XM_017011818.1 597 XM_017011819.1 598 XM_017011820.2 599 XM_017011821.1 600 XM_024446680.1 601 XR_001744581.1 602 XR_002956413.1 603 XR_002956414.1 604 TP53 7157 NM_000546.6 605 NM_001126112.3 606 NM_001126113.3 607 NM_001126114.3 608 NM_001126115.2 609 NM_001126116.2 610 NM_001126117.2 611 NM_001126118.2 612 NM_001276695.3 613 NM_001276696.3 614 NM_001276697.3 615 NM_001276698.3 616 NM_001276699.3 617 NM_001276760.3 618 NM_001276761.3 619 CCNB1 891 NM_031966.4 620 NM_001354844.2 621 NM_001354845.2 622 MAD2L1 4085 NM_002358.4 623 AKT1 207 NM_001382430.1 624 NM_005163.2 625 NM_001014431.2 626 NM_001014432.2 627 NM_001382431.1 628 NM_001382432.1 629 NM_001382433.1 630 XR_002957536.1 631 AKT2 208 NM_001626.6 632 NM_001243027.3 633 NM_001243028.3 634 NM_001330511.1 635 XM_011526614.1 636 XM_011526615.1 637 XM_011526616.1 638 XM_011526618.1 639 XM_011526619.1 640 XM_011526620.1 641 XM_011526622.2 642 XM_017026470.2 643 XM_024451416.1 644 XM_024451417.1 645 AKT3 10000 NM_005465.7 646 NM_181690.2 647 NM_001206729.2 648 NM_001370074.1 649 XM_011544014.2 650 XM_016999985.1 651 XM_024446000.1 652 XM_024446892.1 653 XM_024447938.1 654 PECAM1 5175 NM_000442.5 655 XM_005276880.1 656 XM_005276881.1 657 XM_005276882.1 658 XM_005276883.2 659 XM_011524889.2 660 XM_011524890.1 661 XM_017024738.1 662 XM_017024739.1 663 XM_017024740.1 664 XM_017024741.1 665 KLF5 688 NM_001730.5 666 NM_001286818.2 667 PLXDC1 57125 NM_020405.5 668 UBE3A 7337 NM_130839.5 669 NM_000462.5 670 NM_130838.4 671 NM_001354505.1 672 NM_001354506.2 673 NM_001354507.2 674 NM_001354508.2 675 NM_001354509.2 676 NM_001354511.2 677 NM_001354512.2 678 NM_001354513.2 679 NM_001354523.2 680 NM_001354526.1 681 NM_001354538.2 682 NM_001354539.2 683 NM_001354540.2 684 NM_001354541.2 685 NM_001354542.2 686 NM_001354543.2 687 NM_001354544.2 688 NM_001354545.2 689 NM_001354546.2 690 NM_001354547.2 691 NM_001354548.2 692 NM_001354549.2 693 NM_001354550.2 694 NM_001354551.2 695 NM_001374461.1 696 NR_148916.2 697 XM_011521995.3 698 XM_017022547.2 699 XM_017022548.2 700 XM_017022550.2 701 XM_017022556.2 702 XM_024450043.1 703 RET  5979 NM_020975.6 704 NM_020630.6 705 NM_001355216.1 706 SSX1 6756 NM_005635.4 707 NM_001278691.2 708 SS18 6760 NM_001007559.3 709 NM_005637.4 710 NM_001308201.2 711 XM_006722527.2 712 XM_011526145.1 713 XM_011526147.2 714 XM_011526148.2 715 XM_011526149.2 716 XM_011526150.2 717 XM_011526151.2 718 XM_011526152.2 719 RECQL 5965 NM_002907.4 720 NM_032941.3 721 XM_005253461.3 722 XM_005253462.5 723 XM_005253463.4 724 XM_005253464.4 725 RAN 5901 NM_006325.5 726 NM_001300796.2 727 NM_001300797.2 728 XM_017019772.1 729 XM_017019773.1 730 ABCB1 5243 NM_001348946.2 731 NM_000927.5 732 NM_001348944.2 733 NM_001348945.2 734 ACTB 60 NM_001101.5 735 POSTN 10631 NM_006475.3 736 NM_001135934.2 737 NM_001135935.2 738 NM_001135936.2 739 NM_001286665.2 740 NM_001286666.2 741 NM_001286667.2 742 NM_001330517.2 743 XM_005266232.2 744 XM_017020355.1 745 XM_017020356.1 746 KIF11 3832 NM_004523.4 747 XIAP 331 NM_001167.4 771 NM_001204401.2 772 NM_001378590.1 773 NM_001378591.1 774 NM_001378592.1 775 NR_037916.2 776 NR_165803.1 777 TERT 7015 NM_198253.3 778 NM_001193376.3 779 NR_149162.3 780 NR_149163.3 781 IGF1R 3480 NM_000875.5 782 NM_001291858.2 783 XM_011521516.2 784 XM_011521517.2 785 XM_017022136.1 786 XM_017022137.1 787 XM_017022138.1 788 XM_017022139.1 789 XM_024449913.1 790 MMP9 4318 NM_004994.3 791 CCDC6 8030 NM_005436.5 792 NCOA4 8031 NM_001145263.2 793 NM_005437.4 794 NM_001145260.2 795 NM_001145261.2 796 NM_001145262.2 797 CD44 960 NM_000610.4 798 NM_001001389.2 799 NM_001001390.2 800 NM_001001391.2 801 NM_001001392.2 802 NM_001202555.2 803 NM_001202556.2 804 NM_001202557.2 805 XM_005253231.3 806 XM_005253232.3 807 XM_005253235.3 808 XM_005253238.3 809 XM_005253239.3 810 XM_005253240.3 811 XM_006718388.2 812 XM_006718390.4 813 XM_011520482.2 814 XM_011520483.2 815 XM_011520484.2 816 XM_011520485.2 817 XM_011520486.2 818 XM_011520487.3 819 XM_011520488.2 820 XM_011520489.3 821 XM_017018583.2 822 XM_017018584.2 823 XM_017018585.2 824

Reducing the expression of genes using siRNA-mediated RNAi is also useful in the treatment of viral infections. For example, reducing the expression of a set of genes that enables an infected cell to evade the host's immune system, or a set of genes that is required for viral replication, hinders proliferation of the virus in the host. Such genes, and their mRNA transcripts, are listed in Table 2. Thus, the transcripts of the genes listed in Table 2 are key targets for the treatment of a viral infection using siRNA-mediated RNAi.

Accordingly, in some embodiments, an API useful for the treatment of viral infections in a mammal is siRNA, the siRNA being a double-stranded RNA molecule including an antisense RNA strand and a sense RNA strand, wherein: (a) the antisense RNA strand is 19-29 nucleotides in length and is complementary to contiguous nucleotides in a target mammalian mRNA sequence, the mRNA sequence being selected from the group consisting of SEQ ID NO: 8-21, 482-486, and 748-765, (b) the sense RNA strand is 19-29 nucleotides in length and is complementary to 14-29 nucleotides from the antisense RNA strand, and (c) the double stranded RNA molecule has a double stranded region of 14-29 nucleotides in length and a 3′ overhang region of 0-5 nucleotides in length.

In some embodiments, an API useful for the treatment of viral infections in a mammal is siRNA, the siRNA being a double-stranded RNA molecule including an antisense RNA strand and a sense RNA strand, wherein: (a) the antisense RNA strand is 19-29 nucleotides in length and is complementary to contiguous nucleotides in a target mammalian mRNA sequence, the cDNA sequence being selected from the group consisting of SEQ ID NO: 482-486 and 748-765, (b) the sense RNA strand is 19-29 nucleotides in length and is complementary to 14-29 nucleotides from the antisense RNA strand, and (c) the double stranded RNA molecule has a double stranded region of 14-29 nucleotides in length and a 3′ overhang region of 0-5 nucleotides in length.

TABLE 2 siRNA and shRNA Viral Infection (Virus-Infected Cell) Target Transcripts GenBank GenBank cDNA (cDNA) Gene Gene ID RefSeq Accession SEQ ID NO CD47  961 NM_001777.4   8 NM_198793.3   9 NM_001382306.1  10 XM_005247909.2  11 XM_017007536.1  12 XR_241521.2  13 XR_241522.2  14 XR_924218.2  15 XR_924219.2  16 XR_924220.2  17 XR_001740374.2  18 XR_001740375.2  19 XR_002959610.1  20 XR_002959611.1  21 ACE2 59272 NM_001371415.1 748 NM_021804.3 749 NM_001386259.1 750 NM_001386260.1 751 NM_001388452.1 752 NM_001389402.1 753 CCR5 1234 NM_001394783.1 754 NM_000579.4 755 NM_001100168.2 756 CXCR4 7852 NM_003467.3 482 NM_001008540.2 483 NM_001348056.2 484 NM_001348059.2 485 NM_001348060.2 486 TAT 6898 NM_000353.3 757 PKN2 5586 NM_006256.4 758 NM_001320707.2 759 NM_001320708.2 760 NM_001320709.2 761 XM_011541772.2 762 XM_017001782.2 763 XM_017001783.2 764 EPHA1 2041 NM_005232.5 765

Increased CD47 expression has been observed in fibrotic fibroblast cells, and blocking CD47 reverses fibrosis by increasing phagocytosis of profibrotic fibroblasts and by eliminating suppressive effects on adaptive immunity. In addition to CD47, expression of other the genes listed in Table 3 has been associated with the promotion of fibrosis. Reducing the expression of these genes using siRNA-mediated RNAi is also useful in the treatment of fibrotic diseases. Such genes, and their mRNA transcripts, are listed in Table 3. Thus, the transcripts of the genes listed in Table 3 represent key targets for the treatment of fibrotic diseases using siRNA-mediated RNAi.

In some embodiments, an API useful for the treatment of fibrotic disease in a mammal is siRNA, the siRNA being a double-stranded RNA molecule including an antisense RNA strand and a sense RNA strand, wherein: (a) the antisense RNA strand is 19-29 nucleotides in length and is complementary to contiguous nucleotides in a target mammalian mRNA sequence, the mRNA sequence being selected from the group consisting of SEQ ID NO: 8-21, 40-43, and 766-770, (b) the sense RNA strand is 19-29 nucleotides in length and is complementary to 14-29 nucleotides from the antisense RNA strand, and (c) the double stranded RNA molecule has a double stranded region of 14-29 nucleotides in length and a 3′ overhang region of 0-5 nucleotides in length.

In some embodiments, an API useful for the treatment of fibrotic disease in a mammal is siRNA, the siRNA being a double-stranded RNA molecule including an antisense RNA strand and a sense RNA strand, wherein: (a) the antisense RNA strand is 19-29 nucleotides in length and is complementary to contiguous nucleotides in a target mammalian mRNA sequence, the mRNA sequence being selected from the group consisting of SEQ ID NO: 40-43, and 766-770, (b) the sense RNA strand is 19-29 nucleotides in length and is complementary to 14-29 nucleotides from the antisense RNA strand, and (c) the double stranded RNA molecule has a double stranded region of 14-29 nucleotides in length and a 3′ overhang region of 0-5 nucleotides in length.

TABLE 3 siRNA and shRNA Fibrotic Disease (Fibrotic Cell) Target Transcripts GenBank GenBank cDNA (mRNA) Gene Gene ID RefSeq Accession SEQ ID NO CD47   961 NM_001777.4   8 NM_198793.3   9 NM_001382306.1  10 XM_005247909.2  11 XM_017007536.1  12 XR_241521.2  13 XR_241522.2  14 XR_924218.2  15 XR_924219.2  16 XR_924220.2  17 XR_001740374.2  18 XR_001740375.2  19 XR_002959610.1  20 XR_002959611.1  21 CD274 29126 NM_014143.4  40 NM_001267706.2  41 NM_001314029.2  42 NR_052005.2  43 JUN  3725 NM_002228.4 766 CFTR  1080 NM_000492.4 767 SCNN1A  6337 NM_001038.6 768 NM_001159575.2 769 NM_001159576.2 770

In accordance with some embodiments, the API may be a short hairpin RNA (“shRNA”). Short hairpin RNA is a single-stranded RNA molecule, forming a stem loop structure, that can reduce the expression of a specific gene by causing the degradation of the gene's mRNA transcript(s), which shares partial complementarity with a region of the shRNA molecule. As with siRNA, the process of reducing the expression of a gene using shRNA is referred to as RNAi. See generally, Rao et al. Adv Drug Deliv Rev 61(9):746-59 (2009), which is hereby incorporated by reference herein its entirety.

Several genes have been implicated in promoting cancer progression and cancer cell proliferation through various mechanisms. These genes, and their mRNA transcripts, are listed in Table 1. By reducing the expression of one or more genes from Table 1, cancer progression and cancer cell proliferation may be inhibited. Thus, the transcripts of the genes listed in Table 1 represent key targets for the treatment of cancer using shRNA-mediated RNAi.

Accordingly, in some embodiments, an API useful for the treatment of cancer in a mammal is shRNA, the shRNA being a single-stranded RNA molecule of 44-71 nucleotides in length, and having, in a 5′ to 3′ direction: a first region of 19-29 nucleotides at the 5′ end of the single-stranded RNA molecule, the first region having a first sequence; a second region of 4-11 nucleotides directly adjacent to the first region, the second region having a second sequence; a third region of 19-29 nucleotides directly adjacent to the second region, the third region having a third sequence; and a fourth region of 2 nucleotides at the 3′ end of the single-stranded RNA molecule, directly adjacent to the third region, the fourth region having a fourth sequence, wherein: (a) the first region has the same number of nucleotides as the third region, (b) the third sequence is the reverse-complement of the first sequence, (c) the third region is complementary to contiguous nucleotides in a target mammalian mRNA sequence, the mRNA sequence being selected from the group consisting of SEQ ID NO: 8-747 and 771-824, and (d) the single-stranded RNA molecule is configured to form a stem loop structure, the first region base pairing with the third region to form a stem, the second region forming a loop, and the fourth region forming a 3′ overhang.

In some embodiments, an API useful for the treatment of cancer in a mammal is shRNA, the shRNA being a single-stranded RNA molecule of 44-71 nucleotides in length, and having, in a 5′ to 3′ direction: a first region of 19-29 nucleotides at the 5′ end of the single-stranded RNA molecule, the first region having a first sequence; a second region of 4-11 nucleotides directly adjacent to the first region, the second region having a second sequence; a third region of 19-29 nucleotides directly adjacent to the second region, the third region having a third sequence; and a fourth region of 2 nucleotides at the 3′ end of the single-stranded RNA molecule, directly adjacent to the third region, the fourth region having a fourth sequence, wherein: (a) the first region has the same number of nucleotides as the third region, (b) the third sequence is the reverse-complement of the first sequence, (c) the third region is complementary to contiguous nucleotides in a target mammalian mRNA sequence, the mRNA sequence being selected from the group consisting of SEQ ID NO: 22-747 and 771-824, and (d) the single-stranded RNA molecule is configured to form a stem loop structure, the first region base pairing with the third region to form a stem, the second region forming a loop, and the fourth region forming a 3′ overhang.

In some embodiments, an API useful for the treatment of cancer in a mammal is shRNA, the shRNA being a single-stranded RNA molecule of 44-71 nucleotides in length, and having, in a 5′ to 3′ direction: a first region of 19-29 nucleotides at the 5′ end of the single-stranded RNA molecule, the first region having a first sequence; a second region of 4-11 nucleotides directly adjacent to the first region, the second region having a second sequence; a third region of 19-29 nucleotides directly adjacent to the second region, the third region having a third sequence; and a fourth region of 2 nucleotides at the 3′ end of the single-stranded RNA molecule, directly adjacent to the third region, the fourth region having a fourth sequence, wherein: (a) the first region has the same number of nucleotides as the third region, (b) the third sequence is the reverse-complement of the first sequence, (c) the third region is complementary to contiguous nucleotides in a target mammalian mRNA sequence, the mRNA sequence being selected from the group consisting of SEQ ID NO: 22-37, and (d) the single-stranded RNA molecule is configured to form a stem loop structure, the first region base pairing with the third region to form a stem, the second region forming a loop, and the fourth region forming a 3′ overhang.

In some embodiments, an API useful for the treatment of cancer in a mammal is shRNA, the shRNA being a single-stranded RNA molecule of 44-71 nucleotides in length, and having, in a 5′ to 3′ direction: a first region of 19-29 nucleotides at the 5′ end of the single-stranded RNA molecule, the first region having a first sequence; a second region of 4-11 nucleotides directly adjacent to the first region, the second region having a second sequence; a third region of 19-29 nucleotides directly adjacent to the second region, the third region having a third sequence; and a fourth region of 2 nucleotides at the 3′ end of the single-stranded RNA molecule, directly adjacent to the third region, the fourth region having a fourth sequence, wherein: (a) the first region has the same number of nucleotides as the third region, (b) the third sequence is the reverse-complement of the first sequence, (c) the third region is complementary to contiguous nucleotides in a target mammalian mRNA sequence, the mRNA sequence being selected from the group consisting of SEQ ID NO: 38-39, and (d) the single-stranded RNA molecule is configured to form a stem loop structure, the first region base pairing with the third region to form a stem, the second region forming a loop, and the fourth region forming a 3′ overhang.

In some embodiments, an API useful for the treatment of cancer in a mammal is shRNA, the shRNA being a single-stranded RNA molecule of 44-71 nucleotides in length, and having, in a 5′ to 3′ direction: a first region of 19-29 nucleotides at the 5′ end of the single-stranded RNA molecule, the first region having a first sequence; a second region of 4-11 nucleotides directly adjacent to the first region, the second region having a second sequence; a third region of 19-29 nucleotides directly adjacent to the second region, the third region having a third sequence; and a fourth region of 2 nucleotides at the 3′ end of the single-stranded RNA molecule, directly adjacent to the third region, the fourth region having a fourth sequence, wherein: (a) the first region has the same number of nucleotides as the third region, (b) the third sequence is the reverse-complement of the first sequence, (c) the third region is complementary to contiguous nucleotides in a target mammalian mRNA sequence, the mRNA sequence being selected from the group consisting of SEQ ID NO: 40-43, and (d) the single-stranded RNA molecule is configured to form a stem loop structure, the first region base pairing with the third region to form a stem, the second region forming a loop, and the fourth region forming a 3′ overhang.

In some embodiments, an API useful for the treatment of cancer in a mammal is shRNA, the shRNA being a single-stranded RNA molecule of 44-71 nucleotides in length, and having, in a 5′ to 3′ direction: a first region of 19-29 nucleotides at the 5′ end of the single-stranded RNA molecule, the first region having a first sequence; a second region of 4-11 nucleotides directly adjacent to the first region, the second region having a second sequence; a third region of 19-29 nucleotides directly adjacent to the second region, the third region having a third sequence; and a fourth region of 2 nucleotides at the 3′ end of the single-stranded RNA molecule, directly adjacent to the third region, the fourth region having a fourth sequence, wherein: (a) the first region has the same number of nucleotides as the third region, (b) the third sequence is the reverse-complement of the first sequence, (c) the third region is complementary to contiguous nucleotides in a target mammalian mRNA sequence, the mRNA sequence being selected from the group consisting of SEQ ID NO: 44-51, and (d) the single-stranded RNA molecule is configured to form a stem loop structure, the first region base pairing with the third region to form a stem, the second region forming a loop, and the fourth region forming a 3′ overhang.

Reducing the expression of genes using shRNA-mediated RNAi is also useful in the treatment of viral infections. For example, reducing the expression of a set of genes that enables an infected cell to evade the host's immune system, or a set of genes that is required for viral replication, hinders proliferation of the virus in the host. Such genes, and their mRNA transcripts, are listed in Table 2. Thus, the transcripts of the genes listed in Table 2 are also key targets for the treatment of a viral infection using shRNA-mediated RNAi.

Accordingly, in some embodiments, an API useful for the treatment of viral infections in a mammal is shRNA, the shRNA being a single-stranded RNA molecule of 44-71 nucleotides in length, and having, in a 5′ to 3′ direction: a first region of 19-29 nucleotides at the 5′ end of the single-stranded RNA molecule, the first region having a first sequence; a second region of 4-11 nucleotides directly adjacent to the first region, the second region having a second sequence; a third region of 19-29 nucleotides directly adjacent to the second region, the third region having a third sequence; and a fourth region of 2 nucleotides at the 3′ end of the single-stranded RNA molecule, directly adjacent to the third region, the fourth region having a fourth sequence, wherein: (a) the first region has the same number of nucleotides as the third region, (b) the third sequence is the reverse-complement of the first sequence, (c) the third region is complementary to contiguous nucleotides in a target mammalian mRNA sequence, the mRNA sequence being selected from the group consisting of SEQ ID NO: 8-21, 482-486, and 748-765, and (d) the single-stranded RNA molecule is configured to form a stem loop structure, the first region base pairing with the third region to form a stem, the second region forming a loop, and the fourth region forming a 3′ overhang.

In some embodiments, an API useful for the treatment of viral infections in a mammal is shRNA, the shRNA being a single-stranded RNA molecule of 44-71 nucleotides in length, and having, in a 5′ to 3′ direction: a first region of 19-29 nucleotides at the 5′ end of the single-stranded RNA molecule, the first region having a first sequence; a second region of 4-11 nucleotides directly adjacent to the first region, the second region having a second sequence; a third region of 19-29 nucleotides directly adjacent to the second region, the third region having a third sequence; and a fourth region of 2 nucleotides at the 3′ end of the single-stranded RNA molecule, directly adjacent to the third region, the fourth region having a fourth sequence, wherein: (a) the first region has the same number of nucleotides as the third region, (b) the third sequence is the reverse-complement of the first sequence, (c) the third region is complementary to contiguous nucleotides in a target mammalian mRNA sequence, the mRNA sequence being selected from the group consisting of SEQ ID NO: 482-486 and 748-765, and (d) the single-stranded RNA molecule is configured to form a stem loop structure, the first region base pairing with the third region to form a stem, the second region forming a loop, and the fourth region forming a 3′ overhang.

As noted above, increased CD47 expression has been observed in fibrotic fibroblast cells, and blocking CD47 reverses fibrosis by increasing phagocytosis of profibrotic fibroblasts and by eliminating suppressive effects on adaptive immunity. In addition to CD47, expression of the genes listed in Table 3 has been associated with the promotion of fibrosis. Reducing the expression of these genes using shRNA-mediated RNAi is also useful in the treatment of fibrotic diseases. Such genes, and their mRNA transcripts, are listed in Table 3. Thus, the transcripts of the genes listed in Table 3 also represent key targets for the treatment of fibrotic diseases using shRNA-mediated RNAi.

Accordingly, in some embodiments, an API useful for the treatment of fibrotic disease in a mammal is shRNA, the shRNA being a single-stranded RNA molecule of 44-71 nucleotides in length, and having, in a 5′ to 3′ direction: a first region of 19-29 nucleotides at the 5′ end of the single-stranded RNA molecule, the first region having a first sequence; a second region of 4-11 nucleotides directly adjacent to the first region, the second region having a second sequence; a third region of 19-29 nucleotides directly adjacent to the second region, the third region having a third sequence; and a fourth region of 2 nucleotides at the 3′ end of the single-stranded RNA molecule, directly adjacent to the third region, the fourth region having a fourth sequence, wherein: (a) the first region has the same number of nucleotides as the third region, (b) the third sequence is the reverse-complement of the first sequence, (c) the third region is complementary to contiguous nucleotides in a target mammalian mRNA sequence, the mRNA sequence being selected from the group consisting of SEQ ID NO: 8-21, 40-43, and 766-770, and (d) the single-stranded RNA molecule is configured to form a stem loop structure, the first region base pairing with the third region to form a stem, the second region forming a loop, and the fourth region forming a 3′ overhang.

In some embodiments, an API useful for the treatment of fibrotic disease in a mammal is shRNA, the shRNA being a single-stranded RNA molecule of 44-71 nucleotides in length, and having, in a 5′ to 3′ direction: a first region of 19-29 nucleotides at the 5′ end of the single-stranded RNA molecule, the first region having a first sequence; a second region of 4-11 nucleotides directly adjacent to the first region, the second region having a second sequence; a third region of 19-29 nucleotides directly adjacent to the second region, the third region having a third sequence; and a fourth region of 2 nucleotides at the 3′ end of the single-stranded RNA molecule, directly adjacent to the third region, the fourth region having a fourth sequence, wherein: (a) the first region has the same number of nucleotides as the third region, (b) the third sequence is the reverse-complement of the first sequence, (c) the third region is complementary to contiguous nucleotides in a mammalian mRNA sequence, the mRNA sequence being selected from the group consisting of SEQ ID NO: 40-43 and 766-770, and (d) the single-stranded RNA molecule is configured to form a stem loop structure, the first region base pairing with the third region to form a stem, the second region forming a loop, and the fourth region forming a 3′ overhang.

MicroRNA (“miRNA”)-based therapeutics include miRNAs (and miRNA mimics) and inhibitors of miRNAs (“antimiRs”).

miRNAs are transcribed as single stranded RNA precursors having a stem-loop structure and are subsequently processed in the cytosol by the Dicer enzyme, producing a mature double-stranded product (“mature miRNA product”). These mature miRNA products are thought to have regulatory roles, including RNA silencing and post-transcriptional regulation of gene expression, through their complementarity to mRNA.

Expression of the miRNAs shown in Table 4 is known to be downregulated in various cancers and replenishment of a downregulated miRNA offers a promising therapy for the treatment of cancer.

Accordingly, in some embodiments, an API useful for the treatment of cancer in a mammal is an miRNA selected from the group consisting of SEQ ID NO: 825-844, 849-851, 853, 855, 857, 864, 865, and 867-883.

TABLE 4 Therapeutic Cancer (Cancer Cell) miRNA APIs GenBank cDNA (miRNA) miRNA RefSeq SEQ miRNA Gene Accession ID NO miR-34a MIR34A NR_029610.1 825 miR-34b MIR34B NR_029839.1 826 miR-34c MIR34C NR_029840.1 827 miR-200a MIR200A NR_029834.1 828 miR-200b MIR200B NR_029639.1 829 miR-200c MIR200C NR_029779.1 830 miR-26a-1 MIR26A1 NR_029499.1 831 miR-506 MIR506 NR_030233.1 832 miR-520a MIR520A NR_030189.1 833 miR-520b MIR520B NR_030195.1 834 miR-520c MIR520C NR_030198.1 835 miR-520d MIR520D NR_030204.1 836 miR-520e MIR520E NR_030183.1 837 miR-520f MIR520F NR_030186.1 838 miR-520g MIR520G NR_030206.1 839 miR-520h MIR520H NR_030215.1 840 miR-15a MIR15A NR_029485.1 841 miR-15b MIR15B NR_029663.1 842 miR-16-1 MIR16-1 NR_029486.1 843 miR-16-2 MIR16-2 NR_029525.1 844 miR-Let-7a-1 MIRLET7A1 NR_029476.1 849 (LET7A1) miR-Let-7f-1 MIRLET7F1 NR_029483.1 850 (LET7F1) miR-Let-7d MIRLET7D NR_029481.1 851 (LET7D) miR-31 MIR31 NR_029505.1 853 miR-98 MIR98 NR_029513.1 855 miR-205 MIR205 NR_029622.1 857 miR-324 MIR324 NR_029896.1 864 (MIR324-5P) miR-195 MIR195 NR_029712.1 865 miR-26a-2 MIR26A2 NR_029847.1 867 miR-101-1 MIR101-1 NR_029516.1 868 miR-101-2 MIR101-2 NR_029836.1 869 miR-145 MIR145 NR_029686.1 870 miR-331 MIR331 NR_029895.1 871 miR-29b-1 MIR29B1 NR_029517.1 872 miR-29b-2 MIR29B2 NR_029518.1 873 miR-7-1 MIR7-1 NR_029605.1 874 miR-7-2 MIR7-2 NR_029606.1 875 miR-7-3 MIR7-3 NR_029607.1 876 miR-33a MIR33A NR_029507.1 877 miR-21 MIR21 NR_029493.1 878 miR-203a MIR203A NR_029620.1 879 miR-203b MIR203B NR_039859.1 880 miR-4711 MIR4711 NR_039861.1 881 miR-4689 MIR4689 NR_039838.1 882 miR-122 MIR122 NR_029667.1 883

In contrast to miRNA, an antimiR (also known as an “antagomir”) is a single stranded antisense oligonucleotide (“ASO”) having a sequence that is complementary to the sequence of a region of a target mature miRNA product. Mature miRNA products are short single-stranded RNA molecules that are produced after an miRNA molecule is processed in the cytosol Typically, two mature miRNA products are produced from an miRNA molecule: a 5p RNA molecule (so named because it is processed from the 5′ arm of the duplex formed as the stem of an miRNA), and a 3p RNA molecule (so named because it is processed from the 3′ arm of the duplex formed as the stem of an miRNA). The 5p and 3p molecules may base pair with each other to form a duplex, and each molecule may be functional—and indeed, may serve a separate function-within a cell through their complementarity to mRNA. In some instances, an miRNA molecule is processed such that only a single functional mature miRNA product is produced.

By binding to their target mature miRNA product through complementary base pairing, antimiRs are able to block the mature miRNA product from binding to its target, thereby inhibiting the functioning of the mature miRNA product. Using antimiR to inhibit mature miRNA products is referred to as miRNA knockdown. See generally Quemener et al. Wiley Interdiscip Rev RNA (5):e1594 (2020), which is hereby incorporated by reference herein in its entirety.

antimiRs are 12-25 nucleotides in length and are complementary to consecutive nucleotides of a target mature miRNA product. Different types of nucleic acids may be used to generate an antimiR. Preferably, the antimiR comprises RNA, as RNA/RNA hybrids are very stable. In addition, antimiR may comprise DNA, or comprise both RNA and DNA nucleotides (referred to herein as a “chimera”). An antimiR should bind with high affinity, through complementary base pairing, to the “seed region” of a mature miRNA product, which spans nucleotides 2-8 from the 5′-end of the mature miRNA product (Lennox et al. Gene Therapy 18: 1111-20 (2011), which is hereby incorporated by reference herein in its entirety.)

Over the years, significant improvements in binding affinity, stability, and target modulation effects of antimiRs have been achieved through chemical modifications to the oligonucleotide backbone. An antimiR may therefore be an RNA derivative or a DNA derivative. In some embodiments, the antimiR comprises a modification providing the antimiR with an additional property, for instance resistance to endonucleases and RNaseH, stability (for instance in a bodily fluid), and reduced toxicity. In some embodiments, the modification is a 2′-O-methyl-phosphorothioate oligoribonucleotide modification, a 2′-O-methoxyethyl oligoribonucleotide modification, and combinations thereof. In some embodiments, the antimiR comprises a peptide nucleic acid, locked nucleic acid, or a morpholino phosphorodiamidate. See generally Wahlestedt et al. PNAS 97, 5633-5638 (2000); Elayadi et al. Curr Opin Investig Drugs 2, 558-61 (2001); Larsen et al. Biochim Biophys Acta 1489, 159-166 (1999); Braasch et al. Biochemistry 41, 4503-4510 (2002); Summerton et al. Antisense Nucleic Acid Drug Dev 7, 187-195 (1997), each of which is hereby incorporated by reference herein in its entirety.

Expression of the miRNAs shown in Table 5 is known to be upregulated in various cancers and their mature miRNA products, as shown in Table 6, are preferred targets for miRNA knockdown for the treatment of cancer.

Accordingly, in some embodiments, an API useful for the treatment of cancer in a mammal is an antimiR, the antimiR being a single-stranded nucleic acid molecule of 12-25 nucleotides in length, the antimiR having a sequence of 12-25 contiguous nucleotides that is complementary to contiguous nucleotides in a target mature miRNA product sequence, the mature miRNA product sequence being selected from the group consisting of SEQ ID NO: 884-908, wherein the contiguous nucleotides in the mature miRNA product sequence includes, in a 5′ to 3′ direction, nucleotides 2 to 8 of the mature miRNA product sequence.

TABLE 5 Upregulated miRNA Expression in Cancer miRNA GenBank cDNA (miRNA) Gene RefSeq Accession miRNA SEQ ID NO MIR10B NR_029609.1 miR-10b 845 MIR221 NR_029635.1 miR-221 846 MIR155 NR_030784.1 miR-155 847 MIR630 NR_030359.1 miR-630 848 MIR27A NR_029501.1 miR-27a 852 MIR96 NR_029512.1 miR-96 854 MIR182 NR_029614.1 miR-182 856 MIR93 NR_029510.1 miR-93 858 MIR375 NR_029867.1 miR-375 859 MIR25 NR_029498.1 miR-25 860 MIR106B NR_029831.1 miR-106b 861 MIR512-1 NR_030180.1 miR-512-1 862 MIR512-2 NR_030181.1 miR-512-2 863 MIR18A NR_029488.1 miR-18a 866

TABLE 6 antimiR Cancer (Cancer Cell) Target Mature miRNA Products Mature miRNA (Mature miRNA Product) miRNA Product SEQ ID NO miR-10b hsa-miR-10b-5p 884 hsa-miR-10b-3p 885 miR-221 hsa-miR-221-5p 886 hsa-miR-221-3p 887 miR-155 hsa-miR-155-5p 888 hsa-miR-155-3p 889 miR-630 hsa-miR-630 890 miR-27a hsa-miR-27a-5p 891 hsa-miR-27a-3p 892 miR-96 hsa-miR-96-5p 893 hsa-miR-96-3p 894 miR-182 hsa-miR-182-5p 895 hsa-miR-182-3p 896 miR-93 hsa-miR-93-5p 897 hsa-miR-93-3p 898 miR-375 hsa-miR-375-5p 899 hsa-miR-375-3p 900 miR-25 hsa-miR-25-5p 901 hsa-miR-25-3p 902 miR-106b hsa-miR-106b-5p 903 hsa-miR-106b-3p 904 miR-512-1 hsa-miR-512-5p 905 hsa-miR-512-5p 906 miR-512-2 hsa-miR-512-5p 905 hsa-miR-512-5p 906 miR-18a hsa-miR-18a-5p 907 hsa-miR-18a-3p 908

In other embodiments, an API useful for the treatment of cancer in a mammal is a protein having anti-cancer properties. Anti-cancer properties include inhibiting the proliferation of a cancer cell, inhibiting the proliferation of a tumor, causing the death of a cancer cell, reducing the size of a tumor, or causing the elimination of a tumor. The proteins listed in Table 7 possess anti-cancer properties. Accordingly, a protein having an amino acid sequence selected from the group consisting of SEQ ID NO: 909-929 and homologs thereof is a suitable APIs for use in accordance with embodiments of the invention. In other embodiments, a protein consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 909-929 and homologs thereof is a suitable APIs for use in accordance with embodiments of the invention.

In some embodiments, an API suitable for the treatment of cancer in a mammal is an mRNA encoding an amino acid sequence selected from the group consisting of SEQ ID NO: 909-929 and homologs thereof, the mRNA being configured to be translated in a target cell of the mammal to produce a protein comprising the amino acid sequence. In other embodiments, an API suitable for the treatment of cancer in a mammal is an mRNA encoding an amino acid sequence selected from the group consisting of SEQ ID NO: 909-929 and homologs thereof, the mRNA being configured to be translated in a target cell of the mammal to produce a protein consisting of the amino acid sequence. The mRNA may be codon-optimized for translation in the target cell of the mammal.

TABLE 7 Therapeutic Cancer (Cancer Cell) Protein APIs SEQ Protein ID NO Cyclin-dependent kinase 9 (CDK9) 909 Histidine-rich glycoprotein (HRG) 910 Interferon alpha-2 (IFNA2) 911 Interferon beta (IFNB1) 912 Interferon gamma (IFNG) 913 Interferon lambda-2 (IFNL2) 914 Interferon lambda-3 (IFNL3) 915 Interleukin-27 subunit alpha (IL27) 916 Interleukin-27 subunit beta (EBI3) 917 Guanylate kinase (GUK1) 918 LanC-like protein 2 (LANCL2) 919 GATOR complex protein NPRL2 (NPRL2) 920 Solute carrier family 22 member 2 (SLC22A2) 921 Equilibrative nucleoside transporter 1 922 (SLC29A1) Beclin-1 (BECN1) 923 FK506-binding protein-like (FKBPL) 924 Ribonuclease pancreatic (RNASE1) 925 Probable global transcription activator 926 SNF2L2 (SMARCA2) Metalloproteinase inhibitor 3 (TIMP3) 927 Tumor necrosis factor ligand superfamily 928 member 10 (TNFSF10) Collagen alpha-1(XVIII) chain (COL18A1) 929

Example 1: VSIRPα-siRNA Conjugate Binds Red Blood Cells

FAM-tagged siRNA (FIG. 1A) (SEQ TD NO:965 (sense strand) and SEQ TD NO:966 (antisense strand)) was mixed with vSIRPα at a 1:1 molar ratio (50 pmol of vSIRPα and 50 pmol siRNA). The siRNA was modified at its 3′ end with maleimide. The vSIRPα was designed with cysteine near its C-terminus. Thiol from the cysteine and maleimide were reacted at a neutral pH via click chemistry. The reaction was held overnight in 4° C. shaker, resulting in the vSIRPα-siRNA conjugate shown in FIG. 1A.

The vSIRPα-siRNA conjugate was isolated using a NAP-5 column using the manufacturer's protocol.

To assess the ability of the vSIRPα-siRNA conjugate to bind red blood cells (“RBC”), the vSIRPα-siRNA conjugate was mixed with mouse RBC, as depicted in FIG. 1B. For this experiment, 6×106 red blood cells, in a volume of 2 μl phosphate-buffered saline (“PBS”), were mixed with 2.5 μl of PBS containing the vSIRPα-siRNA conjugate (totaling 50 pmol of the vSIRPα-siRNA conjugate) and 5.5 μl of PBS. A negative control was made by mixing 6×10⁶ red blood cells, in a volume of 2 μl PBS, with 8 μl of PBS. Each mixture was then incubated at room temperature (20-25° C.) for 2 hours (FIG. 1B).

After the incubation, 5 μl of each mixture was diluted 20-fold with PBS, for a total volume of 100 μl for each mixture, and each dilution was placed in a glass-bottom dish and fluorescently imaged at 494-567 nm with a microscope (488 nm excitation wavelength).

As can be seen in FIG. 1C, the negative control shows no fluorescence, whereas the mixture containing the vSIRPα-siRNA conjugate shows a strong fluorescence signal associated with the red blood cells, indicating that the vSIRPα-siRNA conjugate binds RBC.

Example 2: vSIRPα-siRNA Conjugate is Transferred from RBC to Cancer Cells as Demonstrated by Flow Cytometry

500 pmol of the vSIRPα-siRNA conjugate from Example 1 was incubated with 5×10³ mouse RBC in Dulbecco's phosphate-buffered saline (“DPBS”) at a total volume of 20 μl for 30 min at room temperature (20-25° C.).

After 30 min, the mixture was washed with DPBS by centrifugation at 500×g for 10 minutes and the supernatant was removed. The RBC bound with the vSIRPα-siRNA conjugate were then resuspended to 20 μl PBS.

Two cell lines were used, CT26.CL25 (ATCC CRL-2639) and CaCO2 (ATCC HTB-37). CT26.CL25 is a mouse colon carcinoma cell line. CaCO2 is a human colorectal adenocarcinoma cell line that is deficient in expressing CD47, and was thus used as a negative control (Liu et al. J Biol Chem 276(43):40156-66 (2001), which is hereby incorporated by reference herein in its entirety).

Each cell line, grown independently in a cell culture dish, was detached from the dish with trypsin-EDTA. The cells from each dish were then washed with DPBS, counted, and diluted to 10⁵ cells/100 μl PBS. 4 μl of the FAM-vSIRPα-siRNA/RBC resuspension (totaling 10³ RBC and 100 pmol of the vSIRPα-siRNA) was mixed, independently, with each 100 μl dilution of CT26.CL25 cells and CaCO2 cells. Each mixture was incubated for 30 minutes at room temperature (20-25° C.).

After 30 minutes, each mixture was centrifuged, and the supernatants were removed. The cell pellets were resuspended with 200 μl of flow cytometry buffer for flow cytometer analysis.

FIGS. 2A and 2B show flow cytometry results for CaCO2 cells and CT26.CL25 cells, respectively. Flow cytometry was conducted using a 488 nm laser for excitation of the FAM tag, and detected at 525-565 nm wavelength. After the incubation with RBCs bound with FAM-tagged vSIRPα-siRNA, each cancer cell line showed different levels of a shift in florescence. These results show a significant fluorescence shift for CT26.CL25 after incubation with RBC and little shift for CaCO2 after incubation with RBC, indicating that the degree of fluorescence shift is dependent on the level of CD47 on the cancer cells. These results strongly suggest that FAM-tagged vSIRPα-siRNA has been transferred from RBC to CD47 present on the surface of the CT26.CL25 cells.

Example 3: Anti-CD47 Antibody is Transferred from RBC to Cancer Cells as Demonstrated by Flow Cytometry

First, 0.1 μg of Alexa Fluor® 647 anti-mouse CD47 monoclonal antibody (Biolegend, #127510) was incubated with 4×10³ mouse RBC in a total volume of 100 μl DPBS for 30 minutes at room temperature. After incubation, the RBC-antibody mixture was centrifuged at 500 g for 5 minutes. The supernatant was then decanted and the cells resuspended in 20 μl DPBS.

We then mixed 2×10⁵ CT26.CL25 cells (in 100 μl DPBS) with 10 μl of the resuspended RBC-antibody mixture, for a total volume of 110 μl (mixture #1). Similarly, 2×10⁵ CaCO2 cells (in 100 μl DPBS) were mixed with 10 μl of the resuspended RBC-antibody mixture, for a total volume of 110 μl (mixture #2).

Mixture #1 and mixture #2 were incubated for 30 minutes at room temperature. After incubation, 1 ml of DPBS was added to each mixture, followed by centrifugation at 500 g for 5 minutes. For each mixture, after centrifugation, the supernatant was decanted, and the remaining cells were resuspended in 100 μl DPBS and subject to flow cytometry (Beckman, laser 488 nm, detected 650-670 nm).

FIGS. 3A and 3B show these flow cytometry results for CaCO2 cells and CT26.CL25 cells, respectively. Although not as pronounced as the fluorescence shift observed in Example 2, a greater fluorescence shift was observed for CT26.CL25 cells after incubation with RBC compared to the fluorescence shift for CaCO2 cells after incubation with RBC. This, again, indicates that the degree of fluorescence shift is dependent on the level of CD47 on the cancer cells. These results suggest that the Alexa Fluor® 647 anti-mouse CD47 monoclonal antibody was transferred from RBC to CD47 present on the surface of the CT26.CL25 cells.

Example 4: vSIRPα-siRNA Conjugate is Transferred from RBC to Cancer Cells as Demonstrated by Flow Cytometry

The vSIRPα-siRNA-FAM conjugate of Example 1 was further tested for its ability to be transferred from RBC to CD47 present on the surface of CT26.CL25 cells. Experiments were carried out as in Example 2, except that 100 pmol of the conjugate, rather than 500 pmol, was incubated with the RBC.

As shown in the flow cytometry results of FIG. 4 , a significantly greater percentage of CT26.CL25 cells demonstrate FAM fluorescence compared to CaCO2 cells, again, strongly suggesting that FAM-tagged vSIRPα-siRNA has been transferred from RBC to CD47 present on the surface of the CT26.CL25 cells.

Example 5: Cy5.5-Labeled vSIRPα is Transferred from RBC to Cancer Cells as Demonstrated by Flow Cytometry

vSIRPα labeled with Cy5.5 was tested for its ability to be transferred from RBC to CD47 present on the surface of CT26.CL25 cells. Experiments were carried out as in Example 2, except that 200 pmol of the labeled vSIRPα, rather than 500 pmol of conjugate, was incubated with the RBC.

As shown in the flow cytometry results of FIG. 5 , a significantly greater percentage of CT26.CL25 cells demonstrate Cy5.5 fluorescence compared to CaCO2 cells, strongly suggesting that vSIRPα labeled with Cy5.5 has been transferred from RBC to CD47 present on the surface of the CT26.CL25 cells.

Example 6: Anti-CD47 Antibody is Transferred from RBC to Cancer Cells as Demonstrated by Flow Cytometry

An anti-mouse CD47 monoclonal antibody (Biolegend, #127510) labeled with Alexa Flour® 647 was tested for its ability to be transferred from RBC to CD47 present on the surface of CT26.CL25 cells. Experiments were carried out as in Example 3, except that 1 μg of the labeled antibody, rather than 0.1 μg, was incubated with the RBC.

As shown in the flow cytometry results of FIG. 6 , a significantly greater percentage of CT26.CL25 cells demonstrate Alexa Fluor® 647 fluorescence compared to unstained cells, strongly suggesting that the anti-CD47 antibody conjugated to Alexa Flour® 647 has been transferred from RBC to CD47 present on the surface of the CT26.CL25 cells. This experiment also demonstrates that monoclonal antibodies against CD47 are capable of being transferred from RBC to CD47 present on the surface of cancer cells.

Example 7: An Antibody-miR21 Conjugate is Transferred from RBC to Cancer Cells as Demonstrated by Flow Cytometry

An anti-CD47 monoclonal antibody (Bioxcell, #BE0270) conjugated to Cy5 labeled miR21 (SEQ ID NO:878) was tested for its ability to be transferred from RBC to CD47 present on the surface of CT26.CL25 cells. Experiments were carried out as in Example 3, except that 15.8 μg of the antibody conjugate, rather than 0.1 μg, was incubated with the RBC.

As shown in the flow cytometry results of FIG. 7 , a greater percentage of CT26.CL25 cells demonstrate Cy5 fluorescence compared to unstained cells, strongly suggesting that the CD47mAb-miR21-Cy5 conjugate has been transferred from RBC to CD47 present on the surface of the CT26.CL25 cells.

Example 8: Thrombospondin-1 is Transferred from RBC to Cancer Cells as Demonstrated by Flow Cytometry

Thrombospondin-1 (TSP-1) is a matricellular protein that inhibits angiogenesis and endothelial cell proliferation. TSP-1 binds CD47. The TSP-1 signaling pathway has been found to be involved in various conditions, such as renal disease, cardiovascular disease, inflammation, and cancer. The underlying mechanisms and pathways for TSP-1 are yet to be fully elucidated. However, the function of TSP-1 on several key receptors CD36/VEGF and CD47 has been demonstrated. Especially in cancer, the activated TSP-1 and CD47 pathway has been found to reduce tumor growth and metastasis. See Kale et al. Int J Mol Sci, 22(8) (2021) and Kaur et al. J Biol Chem, 285(50), 38923-38932 (2010). Here, we show that a labeled murine TSP-1 is transferred from RBC to the CT26.CL25 cancer cells.

Murine TSP-1 (7859-TH, R&D Systems) labeled with Cy5.5 was tested for its ability to be transferred from RBC to CD47 present on the surface of CT26.CL25 cells. Experiments were carried out as in Example 2, except that 5 μg of Cy5.5 labeled TSP-1, rather than 500 pmol of conjugate, was incubated with the RBC.

As shown in the flow cytometry results of FIG. 8 , a significantly greater percentage of CT26.CL25 cells demonstrate Cy5.5 fluorescence compared to CaCO2 cells, strongly suggesting that Cy5.5-labeled TSP-1 has been transferred from RBC to CD47 present on the surface of the CT26.CL25 cells.

Example 9: vSIRPα-siRNA Conjugate Binds RBC In Vivo

5 nmol of the vSIRPα-siRNA conjugate from Example 1 and 5 nmol of unconjugated siRNA from Example 1 were each stained with the intercalating dye YOYO™_1 Iodide in a molar ratio of 1:1 in a total volume of 120 μl of RNAse-free water. After incubation for 30 minutes at room temperature, the stained conjugate and siRNA were injected into mice as detailed below.

Three Balb/c mice were used for the experiment: one untreated mouse was used as a negative control; one mouse was injected intravenously at the tail vein with 5 nmol of the unconjugated stained siRNA, and one mouse was injected intravenously at the tail vein with 5 nmol of the stained of the vSIRPα-siRNA conjugate.

45 minutes after injection, blood was collected from the mice. 50 μl of whole blood from each sample was washed twice with 1 ml of DPBS by centrifugation at 500×g for 10 minutes and resuspended in DPBS. The resuspended blood was imaged via confocal microscopy using confocal dishes (SPL 100350) and RBC-associated YOYO™-1 Iodide fluorescence signals were compared between different groups (control, siRNA, and conjugate). A 488 nm laser was used (YOYO™-1 Iodide has an excitation wavelength of 491 nm).

As shown in FIG. 9 , RBCs from the mouse injected with the vSIRPα-siRNA conjugate shows fluorescent punctae (arrows of FIG. 9 ), indicating that binding between the vSIRPα-siRNA conjugate and RBCs occurs in vivo.

POTENTIAL CLAIMS

Various embodiments of the present invention may be characterized by the potential claims listed in the paragraphs following this paragraph (and before the actual claims provided at the end of this application). These potential claims form a part of the written description of this application. Accordingly, subject matter of the following potential claims may be presented as actual claims in later proceedings involving this application or any application claiming priority based on this application. Inclusion of such potential claims should not be construed to mean that the actual claims do not cover the subject matter of the potential claims. Thus, a decision to not present these potential claims in later proceedings should not be construed as a donation of the subject matter to the public.

Without limitation, potential subject matter that may be claimed (prefaced with the letter “P” so as to avoid confusion with the actual claims presented below) includes:

P1. A therapeutic compound for RBC-mediated delivery in a mammalian subject to a target cell expressing CD47, the therapeutic compound comprising:

-   -   a CD47-binding protein conjugated to an API so as to form a         conjugate;     -   wherein the CD47-binding protein is selected from the group         consisting of wild type SIRPα (SEQ ID NO: 1), vSIRPα (SEQ ID NO:         3), wild type thrombospondin-1 (TSP-1) (SEQ ID NO: 7), wild type         SIRPγ (SEQ ID NO: 4), vSIRPγ-1 (SEQ ID NO: 5), vSIRPγ-2 (SEQ ID         NO: 6), ALX148 (SEQ ID NO: 962), TTI-661 (SEQ ID NO: 963),         TTI-662 (SEQ ID NO: 964), a homolog of any of the foregoing, and         combinations thereof, and is configured to bind the conjugate to         CD47 of a red blood cell of the subject so as to enable         transport of the conjugate, through the subject's circulatory         system, to the target cell, so that (i) the CD47-binding         protein, being configured to bind the conjugate to the CD47 of         the red blood cell, binds the CD47 of the target cell, thus         transferring the conjugate from the red blood cell to the target         cell so as to form a conjugate-CD47 complex on the target cell,         thereby blocking CD47 and inhibiting CD47 activity as an immune         escape mechanism of the target cell, and (ii) the conjugate is         taken up by the target cell via endocytosis of the         conjugate-CD47 complex, thereby further inhibiting the immune         escape mechanism of the target cell and delivering the API into         the target cell.

P2. A therapeutic compound for RBC-mediated delivery in a mammalian subject to a target cell expressing CD47, the therapeutic compound comprising:

-   -   a CD47-binding protein conjugated to an API so as to form a         conjugate;     -   wherein the CD47-binding protein is selected from the group         consisting of wild type thrombospondin-1 (TSP-1) (SEQ ID NO: 7),         wild type SIRPγ (SEQ ID NO: 4), vSIRPγ-1 (SEQ ID NO: 5),         vSIRPγ-2 (SEQ ID NO: 6), ALX148 (SEQ ID NO: 962), TTI-661 (SEQ         ID NO: 963), TTI-662 (SEQ ID NO: 964), a homolog of any of the         foregoing, and combinations thereof, and is configured to bind         the conjugate to CD47 of a red blood cell of the subject so as         to enable transport of the conjugate, through the subject's         circulatory system, to the target cell, so that (i) the         CD47-binding protein, being configured to bind the conjugate to         the CD47 of the red blood cell, binds the CD47 of the target         cell, thus transferring the conjugate from the red blood cell to         the target cell so as to form a conjugate-CD47 complex on the         target cell, thereby blocking CD47 and inhibiting CD47 activity         as an immune escape mechanism of the target cell, and (ii) the         conjugate is taken up by the target cell via endocytosis of the         conjugate-CD47 complex, thereby further inhibiting the immune         escape mechanism of the target cell and delivering the API into         the target cell.

P3. A therapeutic compound for RBC-mediated delivery in a mammalian subject to a target cell expressing CD47, the therapeutic compound comprising:

-   -   a CD47-binding protein conjugated to an API so as to form a         conjugate;     -   wherein the CD47-binding protein is an anti-CD47 antibody, the         anti-CD47 antibody comprising:     -   (a) a heavy chain variable region including complementarity         determining regions CDR1, CDR2, and CDR3 comprising SEQ ID NO:         932, SEQ ID NO: 933, and SEQ ID NO: 934, respectively, and a         light chain variable region including complementarity         determining regions CDR1, CDR2, and CDR3 comprising SEQ ID NO:         935, SEQ ID NO: 936, and SEQ ID NO: 937, respectively;     -   (b) a heavy chain variable region including complementarity         determining regions CDR1, CDR2, and CDR3 comprising SEQ ID NO:         940, SEQ ID NO: 941, and SEQ ID NO: 942, respectively, and a         light chain variable region including complementarity         determining regions CDR1, CDR2, and CDR3 comprising SEQ ID NO:         943, SEQ ID NO: 944, and SEQ ID NO: 945, respectively;     -   (c) a heavy chain variable region including complementarity         determining regions CDR1, CDR2, and CDR3 comprising SEQ ID NO:         948, SEQ ID NO: 949, and SEQ ID NO: 950, respectively, and a         light chain variable region including complementarity         determining regions CDR1, CDR2, and CDR3 comprising SEQ ID NO:         951, SEQ ID NO: 952, and SEQ ID NO: 953, respectively; or     -   (d) a heavy chain variable region including complementarity         determining regions CDR1, CDR2, and CDR3 comprising SEQ ID NO:         956, SEQ ID NO: 957, and SEQ ID NO: 958, respectively, and a         light chain variable region including complementarity         determining regions CDR1, CDR2, and CDR3 comprising SEQ ID NO:         959, SEQ ID NO: 960, and SEQ ID NO: 961, respectively; and     -   being configured to bind the conjugate to CD47 of a red blood         cell of the subject so as to enable transport of the conjugate,         through the subject's circulatory system, to the target cell, so         that (i) the CD47-binding protein, being configured to bind the         conjugate to the CD47 of the red blood cell, binds the CD47 of         the target cell, thus transferring the conjugate from the red         blood cell to the target cell so as to form a conjugate-CD47         complex on the target cell, thereby blocking CD47 and inhibiting         CD47 activity as an immune escape mechanism of the target cell,         and (ii) the conjugate is taken up by the target cell via         endocytosis of the conjugate-CD47 complex, thereby further         inhibiting the immune escape mechanism of the target cell and         delivering the API into the target cell.

P3.5 The therapeutic compound according to claim P3, wherein the anti-CD47 antibody is a humanized antibody.

P4. The therapeutic compound according to any one of the preceding claims, wherein the CD47-binding protein is conjugated to the API by a bond selected from the group consisting of a covalent bond, a hydrogen bond, an ionic bond, a van der Waals interaction, and combinations thereof.

P5. The therapeutic compound according to any one of the preceding potential claims, wherein the CD47-binding protein is conjugated to the API by a linker.

P6. The therapeutic compound according to claim P5, wherein the linker is cleavable.

P7. The therapeutic compound according to any one of claims P5 and P6, wherein the linker is configured to be cleaved by a lysosomal degradative enzyme.

P8. The therapeutic compound according to any one of the preceding potential claims, wherein the API is selected from the group consisting of RNA, DNA, an RNA derivative, a DNA derivative, a protein, and a small molecule.

P9. The therapeutic compound according to any one of claims P1-P7, wherein the API is selected from the group consisting of siRNA, shRNA, miRNA, antimiR, and mRNA.

P10. The therapeutic compound according to any one of the preceding potential claims, wherein the target cell is a cell selected from the group consisting of a cancer cell, a virus infected cell, a fibrotic cell, and combinations thereof.

P11. The therapeutic compound according to any one of claims P1-P9, wherein the target cell is a cancer cell.

P12. The therapeutic compound according to any one of claims P1-P9, wherein the target cell is a virus-infected cell.

P13. The therapeutic compound according to any one of claims P1-P9, wherein the target cell is a fibrotic cell.

P14. The therapeutic compound according to claim P11, wherein the cancer cell is in a tumor attributable to a cancer selected from the group consisting of brain tumor, spinal cord tumor, retinoblastoma, oral cancer, nasal cavity cancer, paranasal sinus cancer, pharyngeal cancer, laryngeal cancer, neck cancer, head and neck cancer, melanoma, skin cancer, breast cancer, thyroid cancer, malignant adrenal tumor, endocrine cancer, lung cancer, pleural tumor, respiratory tract cancer, esophageal cancer, stomach cancer, small intestine cancer, colon cancer, anal cancer, liver cancer, biliary tract cancer, pancreatic cancer, kidney cancer, bladder cancer, prostate cancer, testicular cancer, penile cancer, cervical cancer, endometrial cancer, choriocarcinoma, ovarian cancer, blood cancer including acute/chronic leukemia, malignant lymphoma and multiple myeloma, bone tumor, soft tissue tumor, childhood leukemia, and childhood cancer.

P15. The therapeutic compound according to claim P11, wherein the cancer cell is attributable to a cancer selected from the group consisting of ovarian serous cystadenocarcinoma, lung adenocarcinoma, cervical and endocervical cancer, head and neck squamous cell carcinoma, thyroid carcinoma, uterine corpus endometrioid carcinoma, prostate adenocarcinoma, mesothelioma, diffuse large B-cell lymphoma, acute leukemia, lung squamous cell carcinoma, acute lymphoblastic leukemia, esophageal carcinoma, myxofibrosarcoma, pancreatic adenocarcinoma, rectum adenocarcinoma, colon adenocarcinoma, acute megakaryoblastic leukemia, breast invasive carcinoma, stomach adenocarcinoma, bladder urothelial carcinoma, cholangiocarcinoma, leukemia, thymic carcinoma, leiomyosarcoma, thymoma, undifferentiated pleomorphic sarcoma, uterine carcinosarcoma, acute myeloid leukemia, glioblastoma multiforme, sarcoma, skin cutaneous melanoma, kidney clear cell carcinoma, dedifferentiated liposarcoma, lymphoma, retinoblastoma, neuroblastoma, osteosarcoma, juvenile myelomonocytic leukemia, gastrointestinal stromal tumor, dysembryoplatic neuroepithelial tumor, adrenocortical cancer, acute leukemia of ambiguous lineage, pheochromocytoma and paraganglioma, glioma, testicular germ cell tumor, supratentorial embryonal tumor NOS, neurofibroma, kidney papillary cell carcinoma, hepatocellular carcinoma, kidney chromophobe, malignant peripheral nerve sheath tumor, ependymoma, adrenocortical carcinoma, nasopharyngeal carcinoma, spindle cells/sclerosing rhabdomyosarcoma, melanoma, choroid plexus carcinoma, undifferentiated spindle cell carcinoma, myoepithelial carcinoma, alveolar rhabdomyosarcoma, rhabdomyosarcoma, atypical teratoid/rhabdoid tumor, desmoplastic small round cell tumor, fibromatosis, synovial sarcoma, wilms tumor, myofibromytosis, fibrolamellar hepatocellular carcinoma, undifferentiated sarcoma NOS, embryonal rhabdomyosarcoma, uveal melanoma, Ewing sarcoma, hepatoblastoma, infantile fibrosarcoma, INI-deficient soft tissue sarcoma NOA, undifferentiated hepatic sarcoma, and medulloblastoma.

P16. The therapeutic compound according to claim P12, wherein the virus-infected cell is infected with a SARS-CoV-2 virus.

P17. The therapeutic compound according to claim P13, wherein the fibrotic cell is associated with cystic fibrosis.

P18. The therapeutic compound according to any one of claims P1-P7, P10, P11, P14, and P15, wherein the API is siRNA, the siRNA being a double-stranded RNA molecule including an antisense RNA strand and a sense RNA strand,

-   -   wherein:     -   (a) the antisense RNA strand is 19-29 nucleotides in length and         is complementary to contiguous nucleotides in a target mammalian         mRNA sequence, the mRNA sequence being selected from the group         consisting of SEQ ID NO: 8-747 and 771-824,     -   (b) the sense RNA strand is 19-29 nucleotides in length and is         complementary to 14-29 nucleotides from the antisense RNA         strand, and     -   (c) the double stranded RNA molecule has a double stranded         region of 14-29 nucleotides in length and a 3′ overhang region         of 0-5 nucleotides in length.

P19. The therapeutic compound according to any one of claims P1-P7, P10, P11, P14, and P15, wherein the API is siRNA, the siRNA being a double-stranded RNA molecule including an antisense RNA strand and a sense RNA strand,

-   -   wherein:     -   (a) the antisense RNA strand is 19-29 nucleotides in length and         is complementary to contiguous nucleotides in a target mammalian         mRNA sequence, the mRNA sequence being selected from the group         consisting of SEQ ID NO: 22-747 and 771-824,     -   (b) the sense RNA strand is 19-29 nucleotides in length and is         complementary to 14-29 nucleotides from the antisense RNA         strand, and     -   (c) the double stranded RNA molecule has a double stranded         region of 14-29 nucleotides in length and a 3′ overhang region         of 0-5 nucleotides in length.

P20. The therapeutic compound according to any one of claims P1-P7, P10, P11, P14, and P15, wherein the API is siRNA, the siRNA being a double-stranded RNA molecule including an antisense RNA strand and a sense RNA strand,

-   -   wherein:     -   (a) the antisense RNA strand is 19-29 nucleotides in length and         is complementary to contiguous nucleotides in a target mammalian         mRNA sequence, the mRNA sequence being selected from the group         consisting of SEQ ID NO: 22-37,     -   (b) the sense RNA strand is 19-29 nucleotides in length and is         complementary to 14-29 nucleotides from the antisense RNA         strand, and     -   (c) the double stranded RNA molecule has a double stranded         region of 14-29 nucleotides in length and a 3′ overhang region         of 0-5 nucleotides in length.

P21. The therapeutic compound according to any one of claims P1-P7, P10, P11, P14, and P15, wherein the API is siRNA, the siRNA being a double-stranded RNA molecule including an antisense RNA strand and a sense RNA strand,

-   -   wherein:     -   (a) the antisense RNA strand is 19-29 nucleotides in length and         is complementary to contiguous nucleotides in a target mammalian         mRNA sequence, the mRNA sequence being selected from the group         consisting of SEQ ID NO: 38-39,     -   (b) the sense RNA strand is 19-29 nucleotides in length and is         complementary to 14-29 nucleotides from the antisense RNA         strand, and     -   (c) the double stranded RNA molecule has a double stranded         region of 14-29 nucleotides in length and a 3′ overhang region         of 0-5 nucleotides in length.

P22. The therapeutic compound according to any one of claims P1-P7, P10, P11, P14, and P15, wherein the API is siRNA, the siRNA being a double-stranded RNA molecule including an antisense RNA strand and a sense RNA strand,

-   -   wherein:     -   (a) the antisense RNA strand is 19-29 nucleotides in length and         is complementary to contiguous nucleotides in a target mammalian         mRNA sequence, the mRNA sequence being selected from the group         consisting of SEQ ID NO: 40-43,     -   (b) the sense RNA strand is 19-29 nucleotides in length and is         complementary to 14-29 nucleotides from the antisense RNA         strand, and     -   (c) the double stranded RNA molecule has a double stranded         region of 14-29 nucleotides in length and a 3′ overhang region         of 0-5 nucleotides in length.

P23. The therapeutic compound according to any one of claims P1-P7, P10, P11, P14, and P15, wherein the API is siRNA, the siRNA being a double-stranded RNA molecule including an antisense RNA strand and a sense RNA strand,

-   -   wherein:     -   (a) the antisense RNA strand is 19-29 nucleotides in length and         is complementary to contiguous nucleotides in a target mammalian         mRNA sequence, the mRNA sequence being selected from the group         consisting of SEQ ID NO: 44-51,     -   (b) the sense RNA strand is 19-29 nucleotides in length and is         complementary to 14-29 nucleotides from the antisense RNA         strand, and     -   (c) the double stranded RNA molecule has a double stranded         region of 14-29 nucleotides in length and a 3′ overhang region         of 0-5 nucleotides in length.

P24. The therapeutic compound according to any one of claims P1-P7, P10, P12, and P16, wherein the API is siRNA, the siRNA being a double-stranded RNA molecule including an antisense RNA strand and a sense RNA strand,

-   -   wherein:     -   (a) the antisense RNA strand is 19-29 nucleotides in length and         is complementary to contiguous nucleotides in a target mammalian         mRNA sequence, the mRNA sequence being selected from the group         consisting of SEQ ID NO: 8-21, 482-486, and 748-765,     -   (b) the sense RNA strand is 19-29 nucleotides in length and is         complementary to 14-29 nucleotides from the antisense RNA         strand, and     -   (c) the double stranded RNA molecule has a double stranded         region of 14-29 nucleotides in length and a 3′ overhang region         of 0-5 nucleotides in length.

P25. The therapeutic compound according to any one of claims P1-P7, P10, P12, and P16, wherein the API is siRNA, the siRNA being a double-stranded RNA molecule including an antisense RNA strand and a sense RNA strand,

-   -   wherein:     -   (a) the antisense RNA strand is 19-29 nucleotides in length and         is complementary to contiguous nucleotides in a target mammalian         mRNA sequence, the mRNA sequence being selected from the group         consisting of SEQ ID NO: 482-486 and 748-765,     -   (b) the sense RNA strand is 19-29 nucleotides in length and is         complementary to 14-29 nucleotides from the antisense RNA         strand, and     -   (c) the double stranded RNA molecule has a double stranded         region of 14-29 nucleotides in length and a 3′ overhang region         of 0-5 nucleotides in length.

P26. The therapeutic compound according to any one of claims P1-P7, P10, P13, and P17, wherein the API is siRNA, the siRNA being a double-stranded RNA molecule including an antisense RNA strand and a sense RNA strand,

-   -   wherein:     -   (a) the antisense RNA strand is 19-29 nucleotides in length and         is complementary to contiguous nucleotides in a target mammalian         mRNA sequence, the mRNA sequence being selected from the group         consisting of SEQ ID NO: 8-21, 40-43, and 766-770,     -   (b) the sense RNA strand is 19-29 nucleotides in length and is         complementary to 14-29 nucleotides from the antisense RNA         strand, and     -   (c) the double stranded RNA molecule has a double stranded         region of 14-29 nucleotides in length and a 3′ overhang region         of 0-5 nucleotides in length.

P27. The therapeutic compound according to any one of claims P1-P7, P10, P13, and P17, wherein the API is siRNA, the siRNA being a double-stranded RNA molecule including an antisense RNA strand and a sense RNA strand,

-   -   wherein:     -   (a) the antisense RNA strand is 19-29 nucleotides in length and         is complementary to contiguous nucleotides in a target mammalian         mRNA sequence, the mRNA sequence being selected from the group         consisting of SEQ ID NO: 40-43 and 766-770,     -   (b) the sense RNA strand is 19-29 nucleotides in length and is         complementary to 14-29 nucleotides from the antisense RNA         strand, and     -   (c) the double stranded RNA molecule has a double stranded         region of 14-29 nucleotides in length and a 3′ overhang region         of 0-5 nucleotides in length.

P28. The therapeutic compound according to any one of claims P1-P7, P10, P11, P14, and P15, wherein the API is shRNA, the shRNA being a single-stranded RNA molecule of 44-71 nucleotides in length, and having, in a 5′ to 3′ direction:

-   -   a first region of 19-29 nucleotides at the 5′ end of the         single-stranded RNA molecule, the first region having a first         sequence;     -   a second region of 4-11 nucleotides directly adjacent to the         first region, the second region having a second sequence;     -   a third region of 19-29 nucleotides directly adjacent to the         second region, the third region having a third sequence; and     -   a fourth region of 2 nucleotides at the 3′ end of the         single-stranded RNA molecule, directly adjacent to the third         region, the fourth region having a fourth sequence,     -   wherein:     -   (a) the first region has the same number of nucleotides as the         third region,     -   (b) the third sequence is the reverse-complement of the first         sequence,     -   (c) the third region is complementary to contiguous nucleotides         in a target mammalian mRNA sequence, the mRNA sequence being         selected from the group consisting of SEQ ID NO: 8-747 and         771-824, and     -   (d) the single-stranded RNA molecule is configured to form a         stem loop structure, the first region base pairing with the         third region to form a stem, the second region forming a loop,         and the fourth region forming a 3′ overhang.

P29. The therapeutic compound according to any one of claims P1-P7, P10, P11, P14, and P15, wherein the API is shRNA, the shRNA being a single-stranded RNA molecule of 44-71 nucleotides in length, and having, in a 5′ to 3′ direction:

-   -   a first region of 19-29 nucleotides at the 5′ end of the         single-stranded RNA molecule, the first region having a first         sequence;     -   a second region of 4-11 nucleotides directly adjacent to the         first region, the second region having a second sequence;     -   a third region of 19-29 nucleotides directly adjacent to the         second region, the third region having a third sequence; and     -   a fourth region of 2 nucleotides at the 3′ end of the         single-stranded RNA molecule, directly adjacent to the third         region, the fourth region having a fourth sequence,     -   wherein:     -   (a) the first region has the same number of nucleotides as the         third region, (b) the third sequence is the reverse-complement         of the first sequence,     -   (c) the third region is complementary to contiguous nucleotides         in a target mammalian mRNA sequence, the mRNA sequence being         selected from the group consisting of SEQ ID NO: 22-747 and         771-824, and     -   (d) the single-stranded RNA molecule is configured to form a         stem loop structure, the first region base pairing with the         third region to form a stem, the second region forming a loop,         and the fourth region forming a 3′ overhang.

P30. The therapeutic compound according to any one of claims P1-P7, P10, P11, P14, and P15, wherein the API is shRNA, the shRNA being a single-stranded RNA molecule of 44-71 nucleotides in length, and having, in a 5′ to 3′ direction:

-   -   a first region of 19-29 nucleotides at the 5′ end of the         single-stranded RNA molecule, the first region having a first         sequence;     -   a second region of 4-11 nucleotides directly adjacent to the         first region, the second region having a second sequence;     -   a third region of 19-29 nucleotides directly adjacent to the         second region, the third region having a third sequence; and     -   a fourth region of 2 nucleotides at the 3′ end of the         single-stranded RNA molecule, directly adjacent to the third         region, the fourth region having a fourth sequence,     -   wherein:     -   (a) the first region has the same number of nucleotides as the         third region, (b) the third sequence is the reverse-complement         of the first sequence,     -   (c) the third region is complementary to contiguous nucleotides         in a target mammalian mRNA sequence, the mRNA sequence being         selected from the group consisting of SEQ ID NO: 22-37, and     -   (d) the single-stranded RNA molecule is configured to form a         stem loop structure, the first region base pairing with the         third region to form a stem, the second region forming a loop,         and the fourth region forming a 3′ overhang.

P31. The therapeutic compound according to any one of claims P1-P7, P10, P11, P14, and P15, wherein the API is shRNA, the shRNA being a single-stranded RNA molecule of 44-71 nucleotides in length, and having, in a 5′ to 3′ direction:

-   -   a first region of 19-29 nucleotides at the 5′ end of the         single-stranded RNA molecule, the first region having a first         sequence;     -   a second region of 4-11 nucleotides directly adjacent to the         first region, the second region having a second sequence;     -   a third region of 19-29 nucleotides directly adjacent to the         second region, the third region having a third sequence; and     -   a fourth region of 2 nucleotides at the 3′ end of the         single-stranded RNA molecule, directly adjacent to the third         region, the fourth region having a fourth sequence,     -   wherein:     -   (a) the first region has the same number of nucleotides as the         third region,     -   (b) the third sequence is the reverse-complement of the first         sequence,     -   (c) the third region is complementary to contiguous nucleotides         in a target mammalian mRNA sequence, the mRNA sequence being         selected from the group consisting of SEQ ID NO: 38-39, and     -   (d) the single-stranded RNA molecule is configured to form a         stem loop structure, the first region base pairing with the         third region to form a stem, the second region forming a loop,         and the fourth region forming a 3′ overhang.

P32. The therapeutic compound according to any one of claims P1-P7, P10, P11, P14, and P15, wherein the API is shRNA, the shRNA being a single-stranded RNA molecule of 44-71 nucleotides in length, and having, in a 5′ to 3′ direction:

-   -   a first region of 19-29 nucleotides at the 5′ end of the         single-stranded RNA molecule, the first region having a first         sequence;     -   a second region of 4-11 nucleotides directly adjacent to the         first region, the second region having a second sequence;     -   a third region of 19-29 nucleotides directly adjacent to the         second region, the third region having a third sequence; and     -   a fourth region of 2 nucleotides at the 3′ end of the         single-stranded RNA molecule, directly adjacent to the third         region, the fourth region having a fourth sequence,     -   wherein:     -   (a) the first region has the same number of nucleotides as the         third region,     -   (b) the third sequence is the reverse-complement of the first         sequence,     -   (c) the third region is complementary to contiguous nucleotides         in a target mammalian mRNA sequence, the mRNA sequence being         selected from the group consisting of SEQ ID NO: 40-43, and     -   (d) the single-stranded RNA molecule is configured to form a         stem loop structure, the first region base pairing with the         third region to form a stem, the second region forming a loop,         and the fourth region forming a 3′ overhang.

P33. The therapeutic compound according to any one of claims P1-P7, P10, P11, P14, and P15, wherein the API is shRNA, the shRNA being a single-stranded RNA molecule of 44-71 nucleotides in length, and having, in a 5′ to 3′ direction:

-   -   a first region of 19-29 nucleotides at the 5′ end of the         single-stranded RNA molecule, the first region having a first         sequence;     -   a second region of 4-11 nucleotides directly adjacent to the         first region, the second region having a second sequence;     -   a third region of 19-29 nucleotides directly adjacent to the         second region, the third region having a third sequence; and     -   a fourth region of 2 nucleotides at the 3′ end of the         single-stranded RNA molecule, directly adjacent to the third         region, the fourth region having a fourth sequence,     -   wherein:     -   (a) the first region has the same number of nucleotides as the         third region,     -   (b) the third sequence is the reverse-complement of the first         sequence,     -   (c) the third region is complementary to contiguous nucleotides         in a target mammalian mRNA sequence, the mRNA sequence being         selected from the group consisting of SEQ ID NO: 44-51, and     -   (d) the single-stranded RNA molecule is configured to form a         stem loop structure, the first region base pairing with the         third region to form a stem, the second region forming a loop,         and the fourth region forming a 3′ overhang.

P34. The therapeutic compound according to any one of claims P1-P7, P10, P12, and P16, wherein the API is shRNA, the shRNA being a single-stranded RNA molecule of 44-71 nucleotides in length, and having, in a 5′ to 3′ direction:

-   -   a first region of 19-29 nucleotides at the 5′ end of the         single-stranded RNA molecule, the first region having a first         sequence;     -   a second region of 4-11 nucleotides directly adjacent to the         first region, the second region having a second sequence;     -   a third region of 19-29 nucleotides directly adjacent to the         second region, the third region having a third sequence; and     -   a fourth region of 2 nucleotides at the 3′ end of the         single-stranded RNA molecule, directly adjacent to the third         region, the fourth region having a fourth sequence,     -   wherein:     -   (a) the first region has the same number of nucleotides as the         third region,     -   (b) the third sequence is the reverse-complement of the first         sequence,     -   (c) the third region is complementary to contiguous nucleotides         in a target mammalian mRNA sequence, the mRNA sequence being         selected from the group consisting of SEQ ID NO: 8-21, 482-486,         and 748-765, and     -   (d) the single-stranded RNA molecule is configured to form a         stem loop structure, the first region base pairing with the         third region to form a stem, the second region forming a loop,         and the fourth region forming a 3′ overhang.

P35. The therapeutic compound according to any one of claims P1-P7, P10, P12, and P16, wherein the API is shRNA, the shRNA being a single-stranded RNA molecule of 44-71 nucleotides in length, and having, in a 5′ to 3′ direction:

-   -   a first region of 19-29 nucleotides at the 5′ end of the         single-stranded RNA molecule, the first region having a first         sequence;     -   a second region of 4-11 nucleotides directly adjacent to the         first region, the second region having a second sequence;     -   a third region of 19-29 nucleotides directly adjacent to the         second region, the third region having a third sequence; and     -   a fourth region of 2 nucleotides at the 3′ end of the         single-stranded RNA molecule, directly adjacent to the third         region, the fourth region having a fourth sequence,     -   wherein:     -   (a) the first region has the same number of nucleotides as the         third region,     -   (b) the third sequence is the reverse-complement of the first         sequence,     -   (c) the third region is complementary to contiguous nucleotides         in a target mammalian mRNA sequence, the mRNA sequence being         selected from the group consisting of SEQ ID NO: 482-486 and         748-765, and     -   (d) the single-stranded RNA molecule is configured to form a         stem loop structure, the first region base pairing with the         third region to form a stem, the second region forming a loop,         and the fourth region forming a 3′ overhang.

P36. The therapeutic compound according to any one of claims P1-P7, P10, P13, and P17, wherein the API is shRNA, the shRNA being a single-stranded RNA molecule of 44-71 nucleotides in length, and having, in a 5′ to 3′ direction:

-   -   a first region of 19-29 nucleotides at the 5′ end of the         single-stranded RNA molecule, the first region having a first         sequence;     -   a second region of 4-11 nucleotides directly adjacent to the         first region, the second region having a second sequence;     -   a third region of 19-29 nucleotides directly adjacent to the         second region, the third region having a third sequence; and     -   a fourth region of 2 nucleotides at the 3′ end of the         single-stranded RNA molecule, directly adjacent to the third         region, the fourth region having a fourth sequence,     -   wherein:     -   (a) the first region has the same number of nucleotides as the         third region,     -   (b) the third sequence is the reverse-complement of the first         sequence,     -   (c) the third region is complementary to contiguous nucleotides         in a target mammalian mRNA sequence, the mRNA sequence being         selected from the group consisting of SEQ ID NO: 8-21, 40-43,         and 766-770, and     -   (d) the single-stranded RNA molecule is configured to form a         stem loop structure, the first region base pairing with the         third region to form a stem, the second region forming a loop,         and the fourth region forming a 3′ overhang.

P37. The therapeutic compound according to any one of claims P1-P7, P10, P13, and P17, wherein the API is shRNA, the shRNA being a single-stranded RNA molecule of 44-71 nucleotides in length, and having, in a 5′ to 3′ direction:

-   -   a first region of 19-29 nucleotides at the 5′ end of the         single-stranded RNA molecule, the first region having a first         sequence;     -   a second region of 4-11 nucleotides directly adjacent to the         first region, the second region having a second sequence;     -   a third region of 19-29 nucleotides directly adjacent to the         second region, the third region having a third sequence; and     -   a fourth region of 2 nucleotides at the 3′ end of the         single-stranded RNA molecule, directly adjacent to the third         region, the fourth region having a fourth sequence,     -   wherein:     -   (a) the first region has the same number of nucleotides as the         third region,     -   (b) the third sequence is the reverse-complement of the first         sequence,     -   (c) the third region is complementary to contiguous nucleotides         in a target mammalian mRNA sequence, the mRNA sequence being         selected from the group consisting of SEQ ID NO: 40-43 and         766-770, and     -   (d) the single-stranded RNA molecule is configured to form a         stem loop structure, the first region base pairing with the         third region to form a stem, the second region forming a loop,         and the fourth region forming a 3′ overhang.

P38. The therapeutic compound according to any one of claims P1-P7, P10, P11, P14, and P15, wherein the API is an miRNA selected from the group consisting of SEQ ID NO: 825-844, 849-851, 853, 855, 857, 864, 865, and 867-883.

P39. The therapeutic compound according to any one of claims P1-P7, P10, P11, P14, and P15, wherein the API is an antimiR, the antimiR being a single-stranded nucleic acid molecule of 12-25 nucleotides in length, the antimiR having a sequence of 12-25 contiguous nucleotides that is complementary to contiguous nucleotides in a target mature miRNA product sequence, the mature miRNA product sequence being selected from the group consisting of SEQ ID NO: 884-908, wherein the contiguous nucleotides in the mature miRNA product sequence includes, in a 5′ to 3′ direction, nucleotides 2 to 8 of the mature miRNA product sequence.

P40. The therapeutic compound according to any one of claims P1-P7, P10, P11, P14, and P15, wherein the API is a small molecule selected from the group consisting of methotrexate; doxorubicin; vinca alkaloids; camptothecin analogues; microtubule-disrupting agents such as auristatins (e.g., MMAE and MMAF) and maytansinoids (e.g., DM1 and DM4); and DNA-damaging agents such as DNA topoisomerase I inhibitors (e.g., SN-38 and exatecan), double-strand break agents (e.g., calicheamicin), cross-linkers (e.g., pyrrolobenzodiazepine dimer-PBD), and alkylators (e.g., duocarmycin and indolinobenzodiazepine dimer-IGN).

P41. The therapeutic compound according to any one of claims P1-P7, P10, P11, P14, and P15, wherein the API is a protein, the protein having an amino acid sequence selected from the group consisting of SEQ ID NO: 909-929 and homologs thereof.

P42. The therapeutic compound according to any one of claims P1-P7, P10, P11, P14, and P15, wherein the API is a protein, the protein consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 909-929 and homologs thereof.

P43. The therapeutic compound according to any one of claims P1-P7, P10, P11, P14, and P15, wherein the API is an mRNA encoding an amino acid sequence selected from the group consisting of SEQ ID NO: 909-929 and homologs thereof, the mRNA being configured to be translated in the target cell to produce a protein comprising the amino acid sequence.

P44. The therapeutic compound according to any one of claims P1-P7, P10, P11, P14, and P15, wherein the API is an mRNA encoding an amino acid sequence selected from the group consisting of SEQ ID NO: 909-929 and homologs thereof, the mRNA being configured to be translated in the target cell to produce a protein consisting of the amino acid sequence.

P45. The therapeutic compound according to any one of claims P43 and P44, wherein the mRNA is codon optimized.

P46. A method of treating cancer in a mammalian subject in need thereof, the method comprising administering a therapeutically effective amount of the therapeutic compound according to any one of claims P1-P1, P14, P15, P18-P23, P28-P33, and P38-P45.

P47. A method of treating viral infection in a mammalian subject in need thereof, the method comprising administering a therapeutically effective amount of the therapeutic compound according to any one of claims P1-P10, P12, P16, P24, P25, P34, and P35.

P48. A method of treating fibrotic disease in a mammalian subject in need thereof, the method comprising administering a therapeutically effective amount of the therapeutic compound according to any one of claims P1-P10, P13, P17, P26, P27, P36, and P37.

P49. The therapeutic compound according to any one of claims P1-P45, wherein the mammalian subject is a human.

P50. The method according to any one of claims P46-P48, wherein the mammalian subject is a human.

P51. A pharmaceutical composition comprising the therapeutic compound according to any one of claims P1-P45 and P49 and a pharmaceutically acceptable carrier.

The embodiments of the invention described above are intended to be merely exemplary; numerous variations and modifications will be apparent to those skilled in the art. All such variations and modifications are intended to be within the scope of the present invention as defined in any appended claims. 

What is claimed is:
 1. A therapeutic compound for RBC-mediated delivery in a mammalian subject to a target cell expressing CD47, the therapeutic compound comprising: a CD47-binding protein conjugated to an API so as to form a conjugate; wherein the CD47-binding protein is selected from the group consisting of wild type SIRPα (SEQ ID NO: 1), vSIRPα (SEQ ID NO: 3), wild type thrombospondin-1 (TSP-1) (SEQ ID NO: 7), wild type SIRPγ (SEQ ID NO: 4), vSIRPγ-1 (SEQ ID NO: 5), vSIRPγ-2 (SEQ ID NO: 6), ALX148 (SEQ ID NO: 962), TTI-661 (SEQ ID NO: 963), TTI-662 (SEQ ID NO: 964), a homolog of any of the foregoing, and combinations thereof, and is configured to bind the conjugate to CD47 of a red blood cell of the subject so as to enable transport of the conjugate, through the subject's circulatory system, to the target cell, so that (i) the CD47-binding protein, being configured to bind the conjugate to the CD47 of the red blood cell, binds the CD47 of the target cell, thus transferring the conjugate from the red blood cell to the target cell so as to form a conjugate-CD47 complex on the target cell, thereby blocking CD47 and inhibiting CD47 activity as an immune escape mechanism of the target cell, and (ii) the conjugate is taken up by the target cell via endocytosis of the conjugate-CD47 complex, thereby further inhibiting the immune escape mechanism of the target cell and delivering the API into the target cell.
 2. The therapeutic compound according to claim 1, wherein the CD47-binding protein is conjugated to the API by a linker.
 3. The therapeutic compound according to claim 2, wherein the linker is cleavable.
 4. The therapeutic compound according to claim 3, wherein the linker is configured to be cleaved by a lysosomal degradative enzyme.
 5. The therapeutic compound according to claim 1, wherein the target cell is a cancer cell.
 6. The therapeutic compound according to claim 5, wherein the API is siRNA, the siRNA being a double-stranded RNA molecule including an antisense RNA strand and a sense RNA strand, wherein: (a) the antisense RNA strand is 19-29 nucleotides in length and is complementary to contiguous nucleotides in a target mammalian mRNA sequence, the mRNA sequence being selected from the group consisting of SEQ ID NO: 22-747 and 771-824, (b) the sense RNA strand is 19-29 nucleotides in length and is complementary to 14-29 nucleotides from the antisense RNA strand, and (c) the double stranded RNA molecule has a double stranded region of 14-29 nucleotides in length and a 3′ overhang region of 0-5 nucleotides in length.
 7. The therapeutic compound according to claim 5, wherein the API is siRNA, the siRNA being a double-stranded RNA molecule including an antisense RNA strand and a sense RNA strand, wherein: (a) the antisense RNA strand is 19-29 nucleotides in length and is complementary to contiguous nucleotides in a target mammalian mRNA sequence, the mRNA sequence being selected from the group consisting of SEQ ID NO: 22-37, (b) the sense RNA strand is 19-29 nucleotides in length and is complementary to 14-29 nucleotides from the antisense RNA strand, and (c) the double stranded RNA molecule has a double stranded region of 14-29 nucleotides in length and a 3′ overhang region of 0-5 nucleotides in length.
 8. The therapeutic compound according to claim 5, wherein the API is siRNA, the siRNA being a double-stranded RNA molecule including an antisense RNA strand and a sense RNA strand, wherein: (a) the antisense RNA strand is 19-29 nucleotides in length and is complementary to contiguous nucleotides in a target mammalian mRNA sequence, the mRNA sequence being selected from the group consisting of SEQ ID NO: 38-39, (b) the sense RNA strand is 19-29 nucleotides in length and is complementary to 14-29 nucleotides from the antisense RNA strand, and (c) the double stranded RNA molecule has a double stranded region of 14-29 nucleotides in length and a 3′ overhang region of 0-5 nucleotides in length.
 9. The therapeutic compound according to claim 5, wherein the API is siRNA, the siRNA being a double-stranded RNA molecule including an antisense RNA strand and a sense RNA strand, wherein: (a) the antisense RNA strand is 19-29 nucleotides in length and is complementary to contiguous nucleotides in a target mammalian mRNA sequence, the mRNA sequence being selected from the group consisting of SEQ ID NO: 40-43, (b) the sense RNA strand is 19-29 nucleotides in length and is complementary to 14-29 nucleotides from the antisense RNA strand, and (c) the double stranded RNA molecule has a double stranded region of 14-29 nucleotides in length and a 3′ overhang region of 0-5 nucleotides in length.
 10. The therapeutic compound according to claim 5, wherein the API is siRNA, the siRNA being a double-stranded RNA molecule including an antisense RNA strand and a sense RNA strand, wherein: (a) the antisense RNA strand is 19-29 nucleotides in length and is complementary to contiguous nucleotides in a target mammalian mRNA sequence, the mRNA sequence being selected from the group consisting of SEQ ID NO: 44-51, (b) the sense RNA strand is 19-29 nucleotides in length and is complementary to 14-29 nucleotides from the antisense RNA strand, and (c) the double stranded RNA molecule has a double stranded region of 14-29 nucleotides in length and a 3′ overhang region of 0-5 nucleotides in length.
 11. The therapeutic compound according to claim 5, wherein the API is shRNA, the shRNA being a single-stranded RNA molecule of 44-71 nucleotides in length, and having, in a 5′ to 3′ direction: a first region of 19-29 nucleotides at the 5′ end of the single-stranded RNA molecule, the first region having a first sequence; a second region of 4-11 nucleotides directly adjacent to the first region, the second region having a second sequence; a third region of 19-29 nucleotides directly adjacent to the second region, the third region having a third sequence; and a fourth region of 2 nucleotides at the 3′ end of the single-stranded RNA molecule, directly adjacent to the third region, the fourth region having a fourth sequence, wherein: (a) the first region has the same number of nucleotides as the third region, (b) the third sequence is the reverse-complement of the first sequence, (c) the third region is complementary to contiguous nucleotides in a target mammalian mRNA sequence, the mRNA sequence being selected from the group consisting of SEQ ID NO: 8-747 and 771-824, and (d) the single-stranded RNA molecule is configured to form a stem loop structure, the first region base pairing with the third region to form a stem, the second region forming a loop, and the fourth region forming a 3′ overhang.
 12. The therapeutic compound according to claim 5, wherein the API is shRNA, the shRNA being a single-stranded RNA molecule of 44-71 nucleotides in length, and having, in a 5′ to 3′ direction: a first region of 19-29 nucleotides at the 5′ end of the single-stranded RNA molecule, the first region having a first sequence; a second region of 4-11 nucleotides directly adjacent to the first region, the second region having a second sequence; a third region of 19-29 nucleotides directly adjacent to the second region, the third region having a third sequence; and a fourth region of 2 nucleotides at the 3′ end of the single-stranded RNA molecule, directly adjacent to the third region, the fourth region having a fourth sequence, wherein: (a) the first region has the same number of nucleotides as the third region, (b) the third sequence is the reverse-complement of the first sequence, (c) the third region is complementary to contiguous nucleotides in a target mammalian mRNA sequence, the mRNA sequence being selected from the group consisting of SEQ ID NO: 22-37, and (d) the single-stranded RNA molecule is configured to form a stem loop structure, the first region base pairing with the third region to form a stem, the second region forming a loop, and the fourth region forming a 3′ overhang.
 13. The therapeutic compound according to claim 5, wherein the API is shRNA, the shRNA being a single-stranded RNA molecule of 44-71 nucleotides in length, and having, in a 5′ to 3′ direction: a first region of 19-29 nucleotides at the 5′ end of the single-stranded RNA molecule, the first region having a first sequence; a second region of 4-11 nucleotides directly adjacent to the first region, the second region having a second sequence; a third region of 19-29 nucleotides directly adjacent to the second region, the third region having a third sequence; and a fourth region of 2 nucleotides at the 3′ end of the single-stranded RNA molecule, directly adjacent to the third region, the fourth region having a fourth sequence, wherein: (a) the first region has the same number of nucleotides as the third region, (b) the third sequence is the reverse-complement of the first sequence, (c) the third region is complementary to contiguous nucleotides in a target mammalian mRNA sequence, the mRNA sequence being selected from the group consisting of SEQ ID NO: 38-39, and (d) the single-stranded RNA molecule is configured to form a stem loop structure, the first region base pairing with the third region to form a stem, the second region forming a loop, and the fourth region forming a 3′ overhang.
 14. The therapeutic compound according to claim 5, wherein the API is shRNA, the shRNA being a single-stranded RNA molecule of 44-71 nucleotides in length, and having, in a 5′ to 3′ direction: a first region of 19-29 nucleotides at the 5′ end of the single-stranded RNA molecule, the first region having a first sequence; a second region of 4-11 nucleotides directly adjacent to the first region, the second region having a second sequence; a third region of 19-29 nucleotides directly adjacent to the second region, the third region having a third sequence; and a fourth region of 2 nucleotides at the 3′ end of the single-stranded RNA molecule, directly adjacent to the third region, the fourth region having a fourth sequence, wherein: (a) the first region has the same number of nucleotides as the third region, (b) the third sequence is the reverse-complement of the first sequence, (c) the third region is complementary to contiguous nucleotides in a target mammalian mRNA sequence, the mRNA sequence being selected from the group consisting of SEQ ID NO: 40-43, and (d) the single-stranded RNA molecule is configured to form a stem loop structure, the first region base pairing with the third region to form a stem, the second region forming a loop, and the fourth region forming a 3′ overhang.
 15. The therapeutic compound according to claim 5, wherein the API is shRNA, the shRNA being a single-stranded RNA molecule of 44-71 nucleotides in length, and having, in a 5′ to 3′ direction: a first region of 19-29 nucleotides at the 5′ end of the single-stranded RNA molecule, the first region having a first sequence; a second region of 4-11 nucleotides directly adjacent to the first region, the second region having a second sequence; a third region of 19-29 nucleotides directly adjacent to the second region, the third region having a third sequence; and a fourth region of 2 nucleotides at the 3′ end of the single-stranded RNA molecule, directly adjacent to the third region, the fourth region having a fourth sequence, wherein: (a) the first region has the same number of nucleotides as the third region, (b) the third sequence is the reverse-complement of the first sequence, (c) the third region is complementary to contiguous nucleotides in a target mammalian mRNA sequence, the mRNA sequence being selected from the group consisting of SEQ ID NO: 44-51, and (d) the single-stranded RNA molecule is configured to form a stem loop structure, the first region base pairing with the third region to form a stem, the second region forming a loop, and the fourth region forming a 3′ overhang.
 16. The therapeutic compound according to claim 5, wherein the API is an miRNA selected from the group consisting of SEQ ID NO: 825-844, 849-851, 853, 855, 857, 864, 865, and 867-883.
 17. The therapeutic compound according to claim 5, wherein the API is an antimiR, the antimiR being a single-stranded nucleic acid molecule of 12-25 nucleotides in length, the antimiR having a sequence of 12-25 contiguous nucleotides that is complementary to contiguous nucleotides in a target mature miRNA product sequence, the mature miRNA product sequence being selected from the group consisting of SEQ ID NO: 884-908, wherein the contiguous nucleotides in the mature miRNA product sequence includes, in a 5′ to 3′ direction, nucleotides 2 to 8 of the mature miRNA product sequence.
 18. The therapeutic compound according to claim 5, wherein the API is a small molecule selected from the group consisting of methotrexate; doxorubicin; vinca alkaloids; camptothecin analogues; microtubule-disrupting agents; and DNA-damaging agents.
 19. The therapeutic compound according to claim 5, wherein the API is a protein, the protein having an amino acid sequence selected from the group consisting of SEQ ID NO: 909-929 and homologs thereof.
 20. The therapeutic compound according to claim 5, wherein the API is an mRNA encoding an amino acid sequence selected from the group consisting of SEQ ID NO: 909-929 and homologs thereof, the mRNA being configured to be translated in the target cell to produce a protein comprising the amino acid sequence. 